FHIT proteins and nucleic acids and methods based thereon

ABSTRACT

The present invention relates to nucleotide sequences of FHIT genes and amino acid sequences of their encoded proteins, as well as derivatives and analogs thereof, and antibodies thereto. The FHIT gene sequence is mutated in diseases involving cell overproliferation, particularly malignancies of the digestive tract. The present invention further relates to the use of FHIT genes and their encoded proteins as diagnostic and therapeutic reagents for the detection and treatment of disease states associated with cell overproliferation.

This is a continuation of U.S. patent application Ser. No. 09/717,054,filed Nov. 21, 2000, which is a continuation of U.S. patent applicationSer. No. 08/605,430, filed Feb. 22, 1996, which issued as U.S. Pat. No.6,242,212 on Jun. 5, 2001, which is a continuation-in-part of U.S.patent application Ser. No. 08/598,873, filed Feb. 9, 1996, which issuedas U.S. Pat. No. 5,928,884 on Jul. 27, 1999, the entire disclosure ofwhich is incorporated herein by reference.

This invention was made in part with government support under Grantnumbers CA51083, CA39860, CA21124, and CA56336 awarded by the NationalCancer Institute. The government has certain rights in the invention.

1. INTRODUCTION

The present invention relates to nucleotide sequences of the tumorsuppressor FHIT genes and amino acid sequences of their encodedproteins, as well as derivatives and analogs thereof and antibodiesthereto. The present invention relates to the use of nucleotidesequences of FHIT genes and amino acid sequences of their encodedproteins, as well as derivatives and analogs thereof and antibodiesthereto, as diagnostic and therapeutic reagents for the detection andtreatment of cancer. The present invention also relates to therapeuticcompositions comprising Fhit proteins, derivatives or analogs thereof,antibodies thereto, nucleic acids encoding the Fhit proteins,derivatives or analogs, and FHIT antisense nucleic acids.

2. BACKGROUND OF THE INVENTION

Cancer remains one of the most severe health problems in America,accounting for substantial fatality and health costs in society.Tumorigenesis in humans is a complex process involving activation ofoncogenes and inactivation of tumor suppressor genes (Bishop, 1991, Cell64:235-248). Tumor suppressor genes in humans have been identifiedthrough studies of genetic changes occurring in cancer cells (Ponder,1990, Trends Genet. 6:213-218; Weinberg, 1991, Science 254:1138-1146).

There is a close association between particular chromosomalabnormalities, e.g., chromosomal translocations, inversions, anddeletions, and certain types of malignancy, indicating that suchabnormalities may have a causative role in the cancer process.Chromosomal abnormalities may lead to gene fusion resulting in chimericoncoproteins, such as is observed in the majority of the tumorsinvolving the myeloid lineage. Alternatively, chromosomal abnormalitiesmay lead to deregulation of protooncogenes by their juxtaposition to aregulatory element active in the hematopoietic cells, such as isobserved in the translocation occurring in the lymphocytic lineage(Virgilio et al., 1993, Proc. Natl. Acad. Sci. USA 90:9275-9279).Deletions may cause loss of tumor suppressor genes, leading tomalignancy.

Nonrandom chromosomal translocations are characteristic of most humanhematopoietic malignancies (Haluska et al., 1987, Ann. Rev. Genet.21:321-345) and may be involved in some solid tumors (Croce, 1987, Cell49:155-156). In B and T cells, chromosomal translocations and inversionsoften occur as a consequence of mistakes during the normal process ofrecombination of the genes for immunoglobulins (Ig) or T-cell receptors(TCR). These rearrangements juxtapose enhancer elements of the Ig or TCRgenes to oncogenes whose expression is then deregulated (Croce, 1987,Cell 49:155-156). In the majority of the cases, the rearrangementsobserved in lymphoid malignancies occur between two differentchromosomes.

The TCL-1 locus on chromosome 14 band q32.1 is frequently involved inthe chromosomal translocations and inversions with the T-cell receptorgenes observed in several post-thymic types of T-cell leukemias andlymphomas, including T-prolymphocytic leukemias (T-PLL) (Brito-Babapulleand Catovsky, 1991, Cancer Genet. Cytogenet. 55:1-9), acute and chronicleukemias associated with the immunodeficiency syndromeataxia-telangiectasia (AT) (Russo et al., 1988, Cell 53:137-144; Russoet al., 1989, Proc. Natl. Acad. Sci. USA 86:602-606), and adult T-cellleukemia (Virgilio et al., 1993, Proc. Natl. Acad. Sci. USA90:9275-9279).

In 1979, a large Italian-American family in Boston was observed to betransmitting a constitutional reciprocal t(3;8)(p14.2;q24) chromosometranslocation (Cohen et al., 1979, N. Engl. J. Med. 301:592-595; Wangand Perkins, 1984, Cancer Genet. Cytogenet. 11:479-481) which segregatedin the family with early onset, bilateral and multifocal clear cellrenal carcinoma (RCC). Follow-up cytogenetic studies in several familialtumors demonstrated that the tumors had lost the derivative 8 chromosomecarrying the translocated 3p14-pter region; consequently, the tumorswere homozygous for all loci telomeric to the 3p14.2 break (Li et al.,1993, Annals of Internal Medicine 118:106-111). It was suggested thatthe translocation affects expression of a tumor suppressor gene (Cohenet al., 1979, N. Engl. J. Med. 301:592-595) and several investigatorshave sought candidate suppressor genes. We had suggested the proteintyrosine phosphatase gamma gene (PTPRG) as a candidate tumor suppressorgene (LaForgia et al., 1991, Proc. Natl. Acad. Sci. USA 88:5036-5040),and that the majority of clear cell RCCs exhibit loss of heterozygosityof a 0.5 Mb region flanking the translocation (Lubinski et al., 1994,Cancer Res. 54:3710-3713; Druck et al., 1995, Cancer Res. 55:5348-5355),although we did not observe aberrations in the remaining PTPRG gene. The3p14.2 region is also included in deletions in numerous other tumortypes, including nasopharyngeal carcinomas (Lo et al., 1994, Int. J.Oncol. 4:1359-1364).

The t(3;8) translocation breakpoint was cloned and a 3 kb transcript ofa candidate tumor suppressor gene was detected using a probe from nearthe breakpoint (Boldog et al., 1993, Proc. Natl. Acad. Sci. USA90:8509-8513); further details concerning this transcript have not beenreported in spite of a later publication from this group relating tothis subject, and reporting a YAC contig of approximately 6 Mb DNAspanning the 3p14.2 3;8 translocation breakpoint (Boldog et al., 1994,Genes, Chromosomes & Cancer 11:216-221). It has also been suggested thatthere may not be a suppressor gene at 3p14.2, that in fact the t(3;8)translocation was a mechanism for losing the von Hippel-Lindau gene, atumor suppressor gene at 3p25 (Gnarra et al., 1994, Nature Genet.7:85-90).

Another cytogenetic landmark in chromosome region 3p14.2 is the mostcommon of the constitutive aphidicolin inducible fragile sites, FRA3B,which is cytogenetically indistinguishable from the t(3;8) translocation(Glover et al., 1988, Cancer Genet. Cytogenet. 31:69-73). Fragile sites,of which over 100 have been described in human (for review, seeSutherland, 1991, Genet. Anal. Tech. Appl. 8:1616-166), are regions ofthe human genome which reveal cytogenetically detectable gaps whenexposed to specific reagents or culture conditions; several folatesensitive, heritable, X-linked and autosomal fragile sites have beenlocalized to unstable CCG or CGG repeats (Yu et al., 1991, Science252:1179-1181; Kremer et al., 1991, Science 252, 1711-1714; Verkerk etal., 1991, Cell 65:905-914; Fu et al., 1991, Cell 67:1047-1058), and forone of these, the FRA11B at 11q23.3, the CCG repeat is within the 5′untranslated region of the CBL2 gene, a known protooncogene (Jones etal., 1995, Nature 376:145-149). Also this fragile site, FRA11B, isassociated with Jacobsen (11q-) syndrome, showing a direct link betweena fragile site and in vivo chromosome breakage (Jones et al., 1994, Hum.Mol. Genet. 3:2123-2130). Because the induced fragile sites resemblegaps or breaks in chromosomes, it has frequently been speculated thatfragile sites could be sites of chromosomal rearrangement in cancer(Yunis and Soreng, 1984, Science 226:1199-1204). Previously identifiedfragile sites have also been shown to be hypermethylated (Knight et al.,1993, Cell 74:127-134); thus methylation of a fragile site in a tumorsuppressor gene regulatory region might cause loss of transcription ofthe suppressor gene, serving as one “hit” in the tumorigenic process, aspointed out previously (Jones et al., 1995, Nature 376:145-149). Theseauthors also suggested that an important contribution of fragile siteexpression in tumorigenesis might be to increase the incidence ofchromosome deletion during tumorigenesis.

The FRA3B region has been delineated by studies of several groups usingrodent-human hybrids; hybrid cells retaining human chromosome 3 or 3 andX, on a hamster background, were treated with aphidicolin or6-thioguanine (to select hybrids which had lost the X chromosome) andsubclones selected. Subclones retaining portions of chromosome 3 withapparent breaks in region 3p14-p21 were characterized for loss orretention of specific 3p markers to determine the position of 3p14-21breaks (LaForgia et al., 1991, Proc. Natl. Acad. Sci. USA 88:5036-5040,LaForgia et al., 1993, Cancer Res. 53:3118-3124; Paradee et al., 1995,Genomics 27:358-361).

Alterations in oncogenes and tumor suppressor genes in small cell lungcancer (SCLC) and non-small cell lung cancer (NSCLC) have beendescribed, the most frequent target being alterations of p53 (Takahashiet al., 1989, Science 246:491-494; Chiba et al., 1990, Oncogene5:1603-1610; Mitsudomi et al., 1992, Oncogene 7:171-180) andretinoblastoma (Harbour et al., 1988, Science 241:353-357; Xu et al.,1994, J. Natl. Cancer Inst. 86:695-699) genes and allelic deletions ofthe short arm of chromosome 3 (3p) (Kok et al., 1987, Nature330:578-581; Naylor et al., 1987, Nature 329:451-454; Rabbitts et al.,1989, Genes Chrom. Cancer 1:95-105). In addition to cytogeneticallyvisible deletions (Whang-Peng et al., 1982, Science 215:181-182; Testaet al., 1994, Genes Chrom. Cancer 11:178-194), loss of heterozygosity(LOH) at loci on 3p has been reported in nearly 100% of SCLC (Kok etal., 1987, Nature 330:578-581; Naylor et al., 1987, Nature 329:451-454;Brauch et al., 1987, N. Engl. J. Med. 317:1109-1113; Yokota et al.,1987, Proc. Natl. Acad. Sci. USA. 84:9252-9256) and in 50% or more ofNSCLC (Brauch et al., 1987, N. Engl. J. Med. 317:1109-1113; Yokota etal., 1987, Proc. Natl. Acad. Sci. USA. 84:9252-9256; Rabbitts et al.,1990, Genes Chrom. Cancer 2:231-238; Hibi et al., 1992, Oncogene7:445-449; Yokoyama et al., 1992, Cancer Res. 52:873-877; Horio et al.,1993, Cancer Res. 53:1-4), strongly suggesting the presence of at leastone tumor suppressor gene in this chromosomal region.

However, the observation that allelic losses often involve most of the3p has hampered the isolation of the involved gene(s). Candidate locihave been identified such as the von-Hippel Lindau gene, located at3p25, which was subsequently found to be rarely mutated in lung cancercell lines (Sekido et al., 1994, Oncogene 9:1599-1604). Other locilocated in a region within 3p21 were reported to be sites of recurrenthomozygous deletions in SCLC (Daly et al., 1993, Oncogene 8:1721-1729;Kok et al., 1993, Proc. Natl. Acad. Sci. USA 90:6071-6075; Kok et al.,1994, Cancer Res. 54:4183-4187). In addition, transfer of subchromosomalfragments of the region 3p21.3-p21.2 to tumor cell lines has suggestedtumor suppressor activity (Killary et al., 1992, Proc. Natl. Acad. Sci.USA 89:10877-10881; Daly et al., 1993, Oncogene 8:1721-1729). Moreproximal deletions in the 3p12-14 region have also been reported(Rabbitts et al., 1989, Genes Chrom. Cancer 1:95-105; Rabbitts et al.,1990, Genes Chrom. Cancer 2:231-238; Daly et al., 1991, Genomics9:113-119).

Lung cancer is a major cause of mortality worldwide and the overallsurvival rate has not improved significantly in the last 20 years.Despite the success achieved by primary prevention, lung cancer is stillan overwhelming medical and social problem. Even in the cohort ofex-smokers lung cancer incidence remains high for several years, as aconsequence of the accumulated damage, and there is an objective needfor strategies aimed at reducing cancer mortality in individuals whohave stopped smoking.

There remains an unfulfilled need to isolate and characterize the genesassociated with digestive tract and other cancers for use as adiagnostic and therapeutic/prophylactic reagent in the detection,treatment, and prevention of such cancers.

Citation of a reference hereinabove shall not be construed as anadmission that such reference is prior art to the present invention.

3. SUMMARY OF THE INVENTION

The present invention relates to nucleotide sequences of PHIT genes, andamino acid sequences of their encoded FHIT proteins, as well asderivatives (e.g., fragments) and analogs thereof, and antibodiesthereto. The present invention further relates to nucleic acidshybridizable to or complementary to the foregoing nucleotide sequencesas well as equivalent nucleic acid sequences encoding a FHIT protein. Ina specific embodiment, the FHIT genes and proteins are human genes andproteins.

Mutations (in particular, deletions) of FHIT gene sequences areassociated with esophageal, gastric, colon, kidney, and other cancers.

The present invention also relates to expression vectors encoding a FHITprotein, derivative or analog thereof, as well as host cells containingthe expression vectors encoding the FHIT protein, derivative or analogthereof. As used herein, “FHIT” shall be used with reference to the FHITgene, whereas “Fhit” shall be used with reference to the protein productof the FHIT gene.

The present invention further relates to the use of nucleotide sequencesof FHIT genes and amino acid sequences of their encoded Fhit proteins asdiagnostic reagents or in the preparation of diagnostic agents useful inthe detection of cancer or precancerous conditions or hyperproliferativedisorders, in particular those associated with chromosomal or molecularabnormalities, in particular at 3p14.2, and/or decreased levels ofexpression, or expression of dysfunctional forms, of the Fhit protein.The invention further relates to the use of nucleotide sequences of FHITgenes and amino acid sequences of their encoded Fhit proteins astherapeutic/prophylactic agents in the treatment/prevention of cancer,in particular, associated with chromosomal or molecular abnormalities at3p14.2, and/or decreased levels of expression, or expression ofdysfunctional forms, of the Fhit protein.

The invention also relates to Fhit derivatives and analogs of theinvention which are functionally active, i.e., they are capable ofdisplaying one or more known functional activities associated with afull-length (wild-type) Fhit protein. Such functional activities includebut are not limited to antigenicity [ability to bind (or compete withFhit for binding) to an anti-Fhit antibody], immunogenicity (ability togenerate antibody which binds to Fhit), and ability to bind (or competewith Fhit for binding) to a receptor/ligand or substrate for Fhit, andability to multmerize with Fhit.

The invention further relates to fragments (and derivatives and analogsthereof) of Fhit which comprise one or more domains of a Fhit protein,e.g., the histadine triad, and/or retain the antigenicity of a Fhitprotein (i.e., are able to be bound by an anti-Fhit antibody).

The FHIT gene and protein sequences disclosed herein, and antibodies tosuch protein sequences, may be used in assays to diagnose cancers, e.g.,digestive tract and airway tumors, associated with chromosomal ormolecular abnormalities at 3p14.2, and/or decreased Fhit protein levelsor activity by detecting or measuring a decrease in FHIT wild-type mRNAfrom a patient sample or by detecting or measuring a decrease in levelsor activity of Fhit protein from a patient sample, or by detecting anaberrant Fhit DNA, mRNA, or protein.

The Fhit protein, or derivatives or analogs thereof, disclosed herein,may be used for the production of anti-Fhit antibodies which antibodiesmay be used diagnostically in immunoassays for the detection ormeasurement of Fhit protein in a patient sample. Anti-Fhit antibodiesmay be used, for example, for the diagnostic detection or measurement ofFhit protein in biopsied cells and tissues.

The present invention also relates to therapeutic compositionscomprising Fhit proteins, derivatives or analogs thereof, antibodiesthereto, and nucleic acids encoding the Fhit proteins, derivatives oranalogs, and FHIT antisense nucleic acids.

The present invention also relates to therapeutic and diagnostic methodsand compositions based on Fhit proteins and nucleic acids. Therapeuticcompounds of the invention include but are not limited to Fhit proteinsand analogs and derivatives (including fragments) thereof; antibodiesthereto; nucleic acids encoding the Fhit proteins, analogs, orderivatives, and FHIT antisense nucleic acids.

The invention provides methods for prevention or treatment of disordersof overproliferation (e.g., cancer and hyperproliferative disorders) byadministering compounds that promote Fhit activity (e.g., Fhit, anagonist of Fhit; nucleic acids that encode Fhit).

The invention also provides methods of prevention and treatment ofdisorders of overproliferation, wherein the patient is hemizygous for adominant-negative FHIT mutation, by administering compounds thatspecifically antagonize the FHIT mutant nucleic acid or protein (e.g.,antibodies or antisense nucleic acids specific to the mutant).

Animal models, diagnostic methods and screening methods forpredisposition to disorders, and methods to identify Fhit agonists andantagonists, are also provided by the invention.

The present invention also relates to methods of production of the Fhitproteins, derivatives and analogs, such as, for example, by recombinantmeans.

In a particular embodiment of the invention described by way of examplein Section 6, a human FHIT sequence is disclosed and shown to be mutatedin various cancers.

4. DESCRIPTION OF THE FIGURES

FIGS. 1A-1B. Organization of the FHIT gene relative to the 3p14.2 FRA3Band translocation sites. A scheme of the normal 3p14.2 region is shown(A) with the chromosomal region (not to scale) represented by the topline with positions of STS markers (position of D3S1234 relative to thegene is not known), the FRA3B represented by the hybrid c13 break andthe t(3;8) translocation break point shown. The dashed portionrepresents the region involved in the homozygous deletions in tumor celllines. Three of the YAC clones used in developing the above markers, mapand cosmid contig are shown with the cosmid contig below and thedistribution of exons in the FHIT transcript shown below the contig.Black and dotted boxes represent coding and noncoding exons,respectively; asterisks indicate exons with start and stop codons. Oneexon (E5) falls within the defined homozygously deleted region. Exons 1(E1), 2 (E2) and 3 (E3) fall centromeric to the t(3;8) translocationbreak and exon 4 (E4) and 6-10 E6-E10) flank the homozygously deletedregion on the centromeric and telomeric sides, respectively.Organization of types of aberrant transcripts from tumor cell lines areillustrated in part B, with zigzag regions representing insertions ofnew sequence, usually repetitive, into the aberrant transcripts. CCL234and 235 are colon carcinoma-derived cell lines in which homozygousdeletion in the fragile region was not detected. In CCL234 RNA, only anabnormal-sized FHIT transcript was detected by RT-PCR amplification andsequencing; the shorter transcript was shown to result from splicing ofexon 3 to exon 5, with omission of the noncoding exon 4, leaving thecoding region intact. With CCL235 RNA as template, apparently normal andaberrant RT-PCR products were amplified, with the aberrant productresulting from splicing of exon 4 to exon 8 with a repetitive insert of140 bp (contributing an in frame Met codon) between E4 and E8. RT-PCRamplification of RNA from HeLa cells, a cervical carcinoma-derived cellline which exhibited a deletion or a rearrangement of DNA near thet(3;8) translocation, revealed normal and aberrant-sized products, thesmallest product resulting from splicing of exon 4 to exon 9. RT-PCRamplification of RNA from KatoIII, a gastric carcinoma-derived cell linewith discontinuous deletions involving the D3S1481 locus and an ˜50 kbpregion between exons 5 and 6, apparently leaving all FHIT exons intact,resulted in only an aberrant-sized product which is missing exons 4through 7, with an 86 bp repeat, inserted downstream of exon 3,contributing an in frame Met codon. Amplification of the RT product fromHT29, a colon carcinoma-derived cell line with a large deletion (−200kbp, about the size of the 648D4 YAC), which included exon 5, gave onlyan aberrant-sized product resulting from splicing of exon 3 to exon 7.Numerous other tumor-derived cell lines from lung carcinoma (1/3tested), osteosarcoma (1/1), NPC (3/3), ovarian carcinoma (2/2), andhematopoietic (4/5) tumors, exhibited aberrant FHIT transcriptionproducts. The RF48 cell line, from a stomach carcinoma without deletion,showed a normal-sized product, as did a lymphoblastoid line with thet(3;8) translocation, a melanoma (WM1158) and a kidney carcinoma(RC17)-derived cell line. Other colon and stomach carcinoma-derivedlines with deletion (AGS, LS180, LoVo), or without deletion (Colo320),showed aberrant reverse transcriptase-polymerase chain reaction (RT-PCR)products (not shown).

FIGS. 2A-2B. Structure of normal and aberrant FHIT cDNAs. The nucleotide(SEQ ID NO:1) and predicted amino acid (SEQ ID NO:2) sequences of theFHIT gene are shown (A) with positions of exons indicated by arrowheadsabove the sequence and positions of primers used in nested PCR and RACEreactions indicated by arrows below the sequence. A schematicpresentation of some of the aberrant transcripts observed in unculturedtumor tissues of digestive organs is shown in B. Only transcripts whichshowed deletion of coding sequence in Table 3 are presented. The topline in B shows the intact FHIT cDNA map. The thick and thin bars showthe coding and untranslated regions, respectively. The positions ofsplice sites are shown by downward arrows, according to the nucleotidenumbers shown above in A. The class I transcripts lack exon 5 whileclass II transcripts retain exon 5 but generally lose exon 8. In thetranscripts with asterisks, insertions of various lengths were observeddownstream of exon 4. E1-10 indicate exons 1-10.

FIGS. 3A-3C. Expression of the FHIT gene in normal tissues and tumors.Northern blot (A, B) and RT-PCR analysis (C) of normal and tumor-derivedFHIT mRNA. Panel A shows a northern blot of normal mRNAs (2 μg/lane)from spleen (lane 1), thymus (lane 2), prostate (lane 3), testis (lane4), ovary (lane 5), small intestine (lane 6), colon (mucosal lining)(lane 7), and peripheral blood leukocytes (lane 8), hybridized with theFHIT cDNA probe. Panel B shows a northern blot of mRNAs (2 μg/lane) fromnormal small intestine (lane 1) and mRNAs from tumor-derived cell lines:KatoIII (lane 2), HK1 (lane 3), LoVo (lane 4), CNE2 (lane 5), CNE1 (lane6), Colo320 (lane 7), LS180 (lane 8), hybridized with the FHIT cDNAprobe (panel B, upper). The same blot was hybridized with a β-actin cDNAprobe (panel B, lower). Panel C shows amplified products observed afternested RT-PCR amplification of mRNAs from matched uncultured tumor (T)and normal (N) tissues of the same patients (J4, 9625, 5586, E37, E32,E3), or mRNAs from tumor tissues only (J9, J7, J3, J1, E3). Arrowheadsshow the positions of amplified products with abnormal DNA sequence. Thedetails of the DNA sequences of corresponding transcripts are shown inTable 2, and FIG. 2B. White dots in the tumor lanes show the position oftranscripts with normal DNA sequence.

FIGS. 4A-4B. (A) Alignment of amino acid sequences of HIT familyproteins and translations of FHIT cDNAs. Alignment was performed usingBOXSHADE version 3.0. Outlining in thick lines indicates two or moreidentical residues at a position; outlining in thin lines indicatessimilarity. The PAPH1 (SEQ ID NO:3) (accession #U32615) and CAPH1 (SEQID NO:4) (accession #U28375) designate the S. pombe and S. cerevisiaediadenosine 5′,5′″-P1, P4 tetraphosphate asymmetric hydrolases (aph1).PHIT (SEQ ID NO:6) indicates the HIT family member from cyanabacterianSynechococcus Sp. (accession #P32084), BHIT (SEQ ID NO:5), the proteinkinase C inhibitor from B. Taurus (bovine; accession #P16436)), MHIT(SEQ ID NO:7) from M. hyorhinis (mycoplasma, accession #M37339), YHIT(SEQ ID NO:8) from S. cerevisiae (accession #Q04344); the Fhit proteinis 69% similar to the S. pombe (PAPH1) gene over a length of 109 aminoacids. (B) In vitro translation products from recombinant plasmidscontaining different alleles of the FHIT gene: pFHIT1 with a deletion ofnoncoding exon 4 (lane 1); pFHIT2 with an insertion of 72 bp betweenexons 4 and 5 (lane 2); pFHIT3 with a wildtype FHIT lacking exon 1 (lane3); the pFHIT full-length wildtype gene in Bluescript (lane 4); controlreaction, in vitro translation from the pBCAH vector, carrying a portionof the extracellular region of the PTPRG gene (predicted molecularweight 40 kDa) (lane 5).

FIG. 5. Organization of the FHIT gene relative to documented chromosomebreaks in the 3p14.2 fragile region. One FHIT allele is disrupted in allthe translocation carriers of the t(3;8) family, with exons 1, 2 and 3remaining on the derivative 3 chromosome and exons-4-10, including theentire coding region, being translocated to the derivative 8 chromosome,as illustrated above. The hybrid cell line, c13, with a de novo FRA3Bbreak just telomeric to exon 5, has lost most of the FHIT coding region.The KatoIII cells apparently retain all FHIT exons but encode only anabnormal transcript which lacks exons 4-7 and thus cannot produce Fhitprotein. The MB436 and HT29 cells have both lost exon 5 through deletionof different segments of the fragile region.

FIG. 6. Hydrophilicity plot of the Fhit deduced protein sequence (SEQ-IDNO:2), plotted using the PEPPLOT program of the Wisconsin GCG softwarefor DNA and protein analysis.

FIGS. 7A-7C. Printout of R50713 nucleotide sequence (SEQ ID NO:9)aligned with the FHIT cDNA sequence (cDNA 7F1) (SEQ ID NO:1), and theR11128 nucleotide sequence (SEQ ID NO:77). The FHIT coding region startsat nucleotide 363 and ends at nucleotide 812.

FIG. 8. Translation in all three reading frames, both 5′ and 3′directions, of the R50713 EST sequence. 5′3′ Frame 1: SEQ ID NOS:10-15and 76; 5′3′ Frame 2: SEQ ID NOS:16-19; 5′3′ Frame 3: SEQ ID NOS:20-25;3′5′ Frame 1: SEQ ID NOS:26-31; 3′5′ Frame 2: SEQ ID NOS:32-36; 3′5′Frame 3: SEQ ID NOS:37-40.

FIG. 9. Translation in all three reading frames, both 5′ and 3′directions, of the R11128 EST sequence. 5′3′ Frame 1: SEQ ID NOS:41-44;5′3′ Frame 2: SEQ ID NOS:45-48; 5′3′ Frame 3: SEQ ID NOS:49-56; 3′5′Frame 1: SEQ ID NOS:57-58; 3′5′ Frame 2: SEQ ID NOS:59-64; 3′5′ Frame 3:SEQ ID NOS:65-68.

FIGS. 10A-10B. (A) Alignment of yeast (S. pombe) Ap4A hydrolase sequence(U32615) (SEQ ID NO:69) with FHIT cDNA (cDNA 7F1) sequence (SEQ IDNO:1). (B) Result of search for homology stretches between U32615 andcDNA 7F1.

FIGS. 11A-11B. Expression of the FHIT gene in small cell lung cancer(SCLC). (A) Expression of the FHIT gene by nested RT-PCR analysis inSCLC tumors (T) and matched normal (N) tissues. Case 83L indicates acell line established from the tumor 83T. Sizes of the amplifiedproducts are shown at the right. (B) A schematic presentation of theaberrant transcripts of types I and II observed in tumor tissue ofSCLCs. The top line shows the intact FHIT cDNA sequence. The thick andthin bars show the coding and untranslated regions, respectively. Thepositions of splice sites are shown by downward arrows, according to thenucleotide numbers. Type I transcripts lack exons 4 to 6, while type IItranscripts lack exons 4 to 8.

FIG. 12A-12D. Expression of the FHIT gene in small cell lung cancer andsequences of FHIT transcripts. (A) FHIT amplified products observedafter nested RT-PCR of mRNA from tumor (T) and normal (N) tissues ofcase 45 and from tumor (T), normal (N) and cell line (L) samples of case83. Arrowheads show the sizes of the amplified products. (B-D) Sequencesof the type I and II abnormal transcripts observed in SCLCs. Arrowsindicate junctions between exons 3 and 4 in the wild-type transcript(WT), between exons 3 and 7 in the abnormal transcripts of type I andbetween exons 3 and 9 in the abnormal transcripts of type II. WTsequence: SEQ ID NO:78. Type I sequence: SEQ ID NO:79. Type II sequence:SEQ ID NO:80.

FIGS. 13A-13G. Expression of the PHIT gene in non small cell lung cancer(NSCLC) and sequences of FHIT transcripts. (A) Expression of the FHITgene by nested RT-PCR analysis in NSCLC tumors (T) and paired normal (N)tissues. Arrowheads indicate the amplified abnormal products. (B-G)Sequences of the abnormal transcripts observed in NSCLC cases 2, 3 and17. Arrows indicate the junctions of exons 4 to 5 in the wild-typeproducts of cases 2 and 17 (2WT, 17WT) and of exon 3 to 4 in the wildtype product of case 3 (3WT). 2A shows the junction between exons 4 and9 in the abnormal product of case 2, 3A shows the junction between exons3 and 8 in the abnormal product of case 3, and 17A shows the junctionbetween exons 4 and 8 in the abnormal product of case 17. WT sequence:SEQ ID NO:81. 3WT sequence: SEQ ID NO:82. 17WT sequence: SEQ ID NO:83.2A sequence: SEQ ID NO:84. 3A sequence: SEQ ID NO:85. 17A sequence: SEQID NO:86.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to nucleotide sequences of FHIT genes andamino acid sequences of their encoded Fhit proteins, as well asderivatives and analogs thereof, and antibodies thereto.

As described by way of example infra, the present inventors haveisolated and characterized a human FHIT gene, that is involved inesophageal, gastric, colon, kidney, and other cancers. Mutations in FHITgene sequences leading to loss of FHIT gene function are associated withcancer.

The present invention further relates to the use of FHIT genes andrelated nucleic acids and their encoded-proteins or derivatives oranalogs thereof, and antibodies thereto, in assays for the detection andin treatment/prevention of disease states associated with chromosomal ormolecular abnormalities and/or increased expression of FHIT, such ascancer. The present invention also relates to therapeutic compositionscomprising Fhit proteins, derivatives or analogs thereof, antibodiesthereto, nucleic acids encoding the Fhit proteins, derivatives oranalogs, and FHIT antisense nucleic acids.

The FHIT gene sequence can be from one of many different species,including but not limited to, vertebrate, mammalian, bovine, ovine,porcine, equine, rodent and human, in naturally occurring-sequence or invariant form, or from any source, whether natural, synthetic, orrecombinant. In a specific embodiment described herein, the FHIT genesequence is a human sequence. The Fhit protein can be that present inone of many different species, including but not limited to, mammalian,bovine, ovine, porcine, equine, rodent and human, in naturally occurringor variant form, or from any source, whether natural, synthetic, orrecombinant. In specific embodiment described herein, the Fhit proteinis a human protein.

As defined herein, a Fhit derivative may be a fragment or amino acidvariant (e.g., an insertion, substitution and/or deletion derivative) ofthe Fhit sequence shown in FIG. 2A as long as the fragment or amino acidvariant is capable of displaying one or more functional activitiesassociated with a full-length Fhit protein. Such functional activitiesinclude but are not limited to antigenicity, i.e., the ability to bindto an anti-Fhit antibody, immunogenicity, i.e., the ability to generatean antibody which is capable of binding a Fhit protein; the ability toinhibit cell proliferation or inhibit tumor growth; the ability to bind(or compete with Fhit for binding) to a substrate for Fhit; ability tomultimerize with Fhit; and, possibly, Ap4A or other diadenosinehydrolase activity. The invention provides fragments of a Fhit proteinconsisting of at least 10 amino acids, or of at least 25 amino acids, orof at least 50 amino acids, or of at least 100 amino acids. Nucleicacids-encoding such derivatives or analogs are also within the scope ofthe invention. A preferred Fhit protein variant is one sharing at least70% amino acid sequence homology, a particularly preferred Fhit proteinvariant is one sharing at least 80% amino acid sequence homology andanother particularly preferred Fhit protein variant is one sharing atleast 90% amino acid sequence homology to the naturally occurring Fhitprotein over at least 25, at least 50, at least 75, at least 100, or atleast 147 (full-length) contiguous amino acids of the FHIT amino acidsequence. As used herein, amino acid sequence homology refers to aminoacid sequences having identical amino acid residues or amino acidsequences containing conservative changes in amino acid residues. Inanother embodiment, a FHIT homologous protein is one that shares theforegoing percentages of sequences identical with the naturallyoccurring FHIT protein over the recited lengths of amino acids. Proteinsencoded by nucleic acids hybridizable to a FHIT gene undernon-stringent, moderately stringent, or stringent conditions are alsoprovided.

The present invention also relates to therapeutic and diagnostic methodsand compositions based on Fhit proteins and nucleic acids and anti-Fhitantibodies. The invention provides for treatment or prevention ofdisorders of overproliferation. (e.g., cancer and hyperproliferativedisorders) by administering compounds that promote Fhit activity (e.g.,Fhit proteins and functionally active analogs and derivatives (includingfragments) thereof; nucleic acids encoding the Fhit proteins, analogs,or derivatives, agonists of Fhit).

The invention also provides methods of treatment or prevention ofdisorders of overproliferation in which the subject is hemizygous for aFhit dominant-negative mutation by administering compounds to thesubject that specifically antagonize, or inhibit, the dominant-negativefunction of the Fhit mutant gene or protein (e.g., antibodies or Fhitantisense nucleic acids specific to the mutant).

Animal models, diagnostic methods and screening methods forpredisposition to disorders are also provided by the invention.

5.1. The FHIT Coding Sequences

FHIT cDNA, genomic sequences and sequences complementary thereto areFHIT nucleic acids provided by the present invention. In a specificembodiment herein, a FHIT cDNA sequence is provided, thus lacking anyintrons. Sequences hybridizable thereto, preferably lacking introns, arealso provided. Nucleic acids comprising FHIT DNA or RNA exon sequencesare also provided; in various embodiments, at least 15, 25 or 50contiguous nucleotides of PHIT exon sequences are in the nucleic acid.Also included within the scope of the present invention are nucleicacids comprising FHIT cDNA or RNA consisting of at least 8 nucleotides,at least 15 nucleotides, at least 25 nucleotides, at least 50nucleotides, at least 100 nucleotides, at least 200 nucleotides, or atleast 350 nucleotides. In various embodiments, nucleic acids areprovided that are less than 2,000, less than 500, less than 275, lessthan 200, less than 100, or less than 50 bases (or bp, ifdouble-stranded). In various embodiments, the nucleic acids are lessthan 300 kb, 200 kb, 100 kb, 50 kb, or 10 kb. Nucleic acids can besingle-stranded or double-stranded. In specific embodiments, isolatednucleic acids are provided that comprise at least 15 contiguousnucleotides of FHIT coding sequences but which do not comprise all or aportion of any FHIT intron. In a specific embodiment, the nucleic acidcomprises at least one FHIT coding exon (exon 5, 6, 7, 8 or 9). Inanother embodiment, the nucleic acid substantially lacks the FHIT intronbetween exon 5 and 6, yet contains exon 5 and at least one other FHITcoding exon selected from among exon 6, exon 7, exon 8, and exon 9. Inyet another embodiment, the nucleic acid comprises at least one FHITexon selected from among exon 1, 2, 3, 4 and 5, and contains at leastone FHIT exon selected from among exon 6, 7, 8, 9 and 10, and ispreferably less than 10 kb in size. In a preferred embodiment the FHITexon sequences appear in the nucleic acid in the order in which theyappear in the genome; in an alternative embodiment, the exon sequencesdo not appear in the same order. In another embodiment, the nucleic acidcomprises all the FHIT exons (exons 1-10) or all the FHIT coding exons(exons 5-9) in contiguous fashion, and thus lacks introns. In yetanother specific embodiment, the nucleic acid comprising FHIT gene exonsequences does not contain sequences of a genomic flanking gene (i.e.,5′ or 3′ to the PHIT gene in the genome). In a specific embodimentherein, a FHIT genomic sequence is provided, thus containing introns.

The invention also provides single-stranded oligonucleotides for use asprimers in PCR that amplify a FHIT sequence-containing fragment, e.g.,an oligonucleotide having the sequence of a hybridizable portion (atleast ˜8 nucleotides) of a FHIT gene, and another oligonucleotide havingthe reverse complement of a downstream sequence in the same strand ofthe FHIT gene, such that each oligonucleotide primes synthesis in adirection toward the other. The oligonucleotides are preferably in therange of 10-35 nucleotides in length.

The full length cDNA sequence for human FHIT is depicted in FIG. 2A (SEQID NO: 1), with the coding region thereof spanning nucleotide numbers1-441 of FIG. 2A. Sequence analysis of the FHIT cDNA of FIG. 2A revealsan open reading frame of 441 nucleotides, encoding a protein of 147amino acids (SEQ ID NO:2).

In accordance with the present invention, any polynucleotide sequencewhich encodes the amino acid sequence of a FHIT gene product can be usedto generate recombinant molecules which direct the expression of Fhit.Included within the scope of the present invention are nucleic acidsconsisting of at least 8 nucleotides that are useful as probes orprimers (i.e., a hybridizable portion) in the detection or amplificationof FHIT.

In a specific embodiment disclosed herein, the invention relates to thenucleic acid sequence of the human FHIT gene. In a preferred, but notlimiting, aspect of the invention, a human FHIT cDNA sequence is thatpresent in plasmid p7F1 as deposited with the ATCC and assigned ATCCAccession Number 69977. Such a sequence can be cloned and sequenced, forexample, as described in Section 6, infra. The invention also relates tonucleic acid sequences hybridizable or complementary to the foregoingsequences or equivalent to the foregoing-sequences in that theequivalent nucleic acid sequences also encode a protein productdisplaying Fhit functional activity.

Nucleic acids encoding fragments and derivatives of FHIT areadditionally described infra.

The invention also relates to nucleic acids hybridizable to orcomplementary to the above-described nucleic acids comprising FHITsequences. In specific aspects, nucleic acids are provided whichcomprise a sequence complementary to at least 10, 25, 50, 100, or 200nucleotides or the entire coding region of a FHIT gene. In a specificembodiment, a nucleic acid which is hybridizable to a FHIT nucleic acid,or to a nucleic acid encoding a Fhit derivative, under conditions of lowstringency is provided. By way of example and not limitation, proceduresusing such conditions of low stringency are as follows (see also Shiloand Weinberg, 1981, Proc. Natl. Acad. Sci. USA 78:6789-6792): Filterscontaining DNA are pretreated for 6 h at 40° C. in a solution containing35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1%Ficoll, 1% BSA, and 500 μg/ml denatured salmon sperm DNA. Hybridizationsare carried out in the same solution with the following modifications:0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg/ml salmon sperm DNA, 10%(wt/vol) dextran sulfate, and 5-20×10⁶ cpm ³²P-labeled probe is used.Filters are incubated in hybridization mixture for 18-20 h at 40° C.,and then washed for 1.5 h at 55° C. in a solution containing 2×SSC, 25mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS. The wash solution isreplaced with fresh solution and incubated an additional 1.5 h at 60° C.Filters are blotted dry and exposed for autoradiography. If necessary,filters are washed for a third time at 65-68° C. and reexposed to filmother conditions of low stringency which may be used are well known inthe art (e.g., as employed for cross-species hybridizations).

In another specific embodiment, a nucleic acid which is hybridizable toa FHIT nucleic acid under conditions of high stringency is provided (seeinfra).

In a preferred aspect, polymerase chain reaction (PCR) is used toamplify a desired nucleic acid sequence in a library or from a tissuesource by using oligonucleotide primers representing known FHITsequences. Such primers may be used to amplify sequences of interestfrom an RNA or DNA source, preferably a cDNA library. PCR can be carriedout, e.g., by use of a Perkin-Elmer Cetus thermal cycler and Taqpolymerase (Gene Amp™). The DNA being amplified can include mRNA or cDNAor genomic DNA from any eukaryotic species. One can choose to synthesizeseveral different degenerate primers, for use in the PCR reactions. Itis also possible to vary the stringency of hybridization conditions usedin priming the PCR reactions, to allow for greater or lesser degrees ofnucleotide sequence homology between the FHIT gene being cloned and theknown FHIT gene. Other means for primer dependent amplification ofnucleic acids are known to those of skill in the art and can be used.

After successful amplification of a segment of a FHIT gene (e.g., anallelic or polymorphic variant or species homolog of a known FHIT gene)that segment may be molecularly cloned and sequenced, and utilized as aprobe to isolate a complete cDNA or genomic clone. This, in turn, willpermit the determination of the gene's complete nucleotide sequence, theanalysis of its expression, and the production of its protein productfor functional analysis, as described infra. In this fashion, additionalgenes encoding Fhit proteins may be identified. Alternatively, the FHITgene of the present invention may be isolated through an exon trappingsystem, using genomic DNA (Nehls et al., 1994, Oncogene 9(8):2169-2175;Verna et al., 1993, Nucleic Acids Res. 21(22):5198:5202; Auch et al.,1990, Nucleic Acids Res. 18(22):6743-6744).

Potentially, any eukaryotic cell can serve as the nucleic acid sourcefor the molecular cloning of the FHIT gene. The nucleic acid sequencesencoding FHIT can be isolated from, for example, human, porcine, bovine,feline, avian, equine, canine, rodent, as well as additional primatesources. The DNA may be obtained by standard procedures known in the artfrom, for example, cloned DNA (e.g., a DNA “library”), by chemicalsynthesis, by cDNA cloning, or by the cloning of genomic DNA, orfragments thereof, purified from a desired cell. (See, for example,Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2d Ed.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Glover,D. M. (ed.), 1985, DNA Cloning: A Practical Approach, MRL Press, Ltd.,Oxford, U.K. Vol. I, II.) Clones derived from genomic DNA may containregulatory and intron DNA regions in addition to coding regions whileclones derived from cDNA will contain only FHIT exon sequences. Whateverthe source, the gene should be molecularly cloned into a suitable vectorfor propagation of the gene. In a particular embodiment, a preferredsource of nucleic acid for the isolation of FHIT gene sequences is fromkidney or stomach or lung cells.

In the molecular cloning of the gene from genomic DNA, DNA fragments aregenerated, some of which will encode the desired gene. The DNA may becleaved at specific sites using various restriction enzymes.Alternatively, one may use DNAse in the presence of manganese tofragment the DNA, or the DNA can be physically sheared, as for example,by sonication. The linear DNA fragments can then be separated accordingto size by standard techniques, including but not limited to, agaroseand polyacrylamide gel electrophoresis and column chromatography.

Once the DNA fragments are generated, identification of the specific DNAfragment containing the desired gene may be accomplished in a number ofways. For example, a FHIT gene of the present invention or its specificRNA, or a fragment thereof, such as a probe or primer, may be isolatedand labeled and then used in hybridization assays to detect a generatedFHIT gene (Benton, W. and Davis, R., 1977, Science 196:180; Grunstein,M. And Hogness, D., 1975, Proc. Natl. Acad. Sci. USA 72:3961). Those DNAfragments sharing substantial sequence homology to the probe willhybridize under high stringency conditions. The phrase “high stringencyconditions” as used herein refers to those hybridizing conditions that(1) employ low ionic strength and high temperature for washing, forexample, 0.015 M NaCl/0.0015 M sodium citrate/0.1% SDS at 50° C.; (2)employ during hybridization a denaturing agent such as formamide, forexample, 50% (vol/vol) formamide with 0.1% bovine serum albumin/0.1%Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5with 750 mM NaCl, 75 mM sodium citrate at 42° C.; or (3) employ 50%formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50 g/ml), 0.1% SDS, and10% dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC and 0.1%SDS.

It is also possible to identify the appropriate fragment by restrictionenzyme digestion(s) and comparison of fragment sizes with those expectedaccording to a known restriction map. Further selection can be carriedout on the basis of the properties of the gene. Alternatively, thepresence of the gene may be detected by assays based on the physical,chemical, or immunological properties of its expressed product. Forexample, cDNA clones, or genomic DNA clones which hybrid-select theproper mRNAs, can be selected which produce a protein that has similaror identical electrophoretic migration, isolectric focusing behavior,proteolytic digestion maps, binding activity or antigenic properties asknown for FHIT. Alternatively, the FHIT protein may be identified bybinding of labeled antibody to the putatively FHIT expressing clones,e.g., in an ELISA (enzyme-linked immunosorbent assay)-type procedure.

The FHIT gene can also be identified by mRNA selection by nucleic acidhybridization followed by in vitro translation. In this procedure,fragments are used to isolate complementary mRNAs by hybridization. SuchDNA fragments may represent available, purified FHIT DNA of another FHITgene.

Immunoprecipitation analysis or functional assays of the in vitrotranslation products of the isolated products of the isolated mRNAsidentifies the mRNA and, therefore, the complementary DNA fragments thatcontain the desired sequences. In addition, specific mRNAs may beselected by adsorption of polysomes isolated from cells to immobilizedantibodies specifically directed against FHIT protein. A radiolabelledFHIT cDNA can be synthesized using the selected mRNA (from the adsorbedpolysomes) as a template. The radiolabelled mRNA or cDNA may then beused as a probe to identify the FHIT DNA fragments from among othergenomic DNA fragments.

Alternatives to isolating the FHIT genomic DNA include, but are notlimited to, chemically synthesizing the gene sequence itself from aknown sequence or making cDNA to the mRNA which encodes the FHITprotein. For example, RNA useful in cDNA cloning of the FHIT gene can beisolated from cells which express FHIT, e.g., kidney or stomach or lungcells. Other methods are known to those of skill in the art and arewithin the scope of the invention.

The identified and isolated gene can then be inserted into anappropriate cloning vector. A large number of vector-host systems knownin the art may be used. Possible vectors include, but are not limitedto, plasmids or modified viruses, but the vector system must becompatible with the host cell used. Such vectors include, but are notlimited to, bacteriophages such as lambda derivatives, or plasmids suchas PBR322 or pUC plasmid derivatives or the Bluescript vector(Stratagene). The insertion into a cloning vector can, for example, beaccomplished by ligating the DNA fragment into a cloning vector whichhas complementary cohesive termini. However, if the complementaryrestriction sites used to fragment the DNA are not present in thecloning vector, the ends of the DNA molecules may be enzymaticallymodified. Alternatively, any site desired may be produced by ligatingnucleotide sequences (linkers) onto the DNA termini; these ligatedlinkers may comprise specific chemically synthesized oligonucleotidesencoding restriction endonuclease recognition sequences. In analternative method, the cleaved vector and FHIT gene may be modified byhomopolymeric tailing. Recombinant molecules can be introduced into hostcells via transformation, transfection, infection, electroporation, orother methods known to those of skill in the art, so that many copies ofthe gene sequence are generated.

In an alternative method, the desired gene may be identified andisolated after insertion into a suitable cloning vector in a “shot gun”approach. Enrichment for the desired gene, for example, by sizefractionization, can be done before insertion into the cloning vector.

In specific embodiments, transformation of host cells with recombinantDNA molecules that incorporate the isolated FHIT gene, cDNA, orsynthesized DNA sequence enables is generation of multiple copies of thegene. Thus, the gene may be obtained in large quantities by growingtransformants, isolating the recombinant DNA molecules from thetransformants and, when necessary, retrieving the inserted gene from theisolated recombinant DNA.

Oligonucleotides containing a portion of the FHIT coding or non-codingsequences, or which encode a portion of the FHIT protein (e.g., primersfor use in PCR) can be synthesized by standard methods commonly known inthe art. Such oligonucleotides preferably have a size in the range of 8to 25 nucleotides. In a specific embodiment herein, sucholigonucleotides have a size in the range of 15 to 25 nucleotides or 15to 35 nucleotides.

The FHIT sequences provided by the instant invention include thosenucleotide sequences encoding substantially the same amino acidsequences as found in native Fhit proteins, and those encoded amino acidsequences with functionally equivalent amino acids, as well as thoseencoding other Fhit derivatives or analogs, as described infra for Fhitderivatives and analogs.

5.2. Expression of the FHIT Gene

In accordance with the present invention, nucleotide sequences codingfor a FHIT protein, derivative, e.g. fragment, or analog thereof, can beinserted into an appropriate expression vector, i.e., a vector whichcontains the necessary elements for the transcription and translation ofthe inserted protein-coding sequence, for the generation of recombinantDNA molecules that direct the expression of a FHIT protein. Such PHITpolynucleotide sequences, as well as other polynucleotides or theircomplements, may also be used in nucleic acid hybridization assays,Southern and Northern blot analysis, etc. In a specific embodiment, ahuman FHIT gene, or a sequence encoding a functionally active portion ofa human FHIT gene is expressed. In yet another embodiment, a derivativeor fragment of a human FHIT gene is expressed.

Due to the inherent degeneracy of the genetic code, other DNA sequenceswhich encode substantially the same or a functionally equivalent FHITamino acid sequence, is within the scope of the invention. Such DNAsequences include those which are capable of hybridizing to the humanFHIT sequence under stringent conditions.

Altered DNA sequences which may be used in accordance with the inventioninclude deletions, additions or substitutions of different nucleotideresidues resulting in a sequence that encodes the same or a functionallyequivalent gene product. The gene product itself may contain deletions,additions or substitutions of amino acid residues within an FHITsequence, which result in a silent change thus producing a functionallyequivalent FHIT protein. Such amino acid substitutions may be made onthe basis of similarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues involved.For example, negatively charged amino acids include aspartic acid andglutamic acid; positively charged amino acids include lysine andarginine; amino acids with uncharged polar head groups having similarhydrophilicity values include the following: leucine, isoleucine,valine; glycine, alanine; asparagine, glutamine; serine, threonine;phenylalanine, tyrosine.

The DNA sequences of the invention may be engineered in order to alter aFHIT coding sequence for a variety of ends including but not limited toalterations which modify processing and expression of the gene product.For example, mutations may be introduced using techniques which are wellknown in the art, e.g., site-directed mutagenesis, to insert newrestriction sites, etc.

In another embodiment of the invention, a FHIT gene sequence or aderivative thereof is ligated to a non-FHIT sequence to encode achimeric fusion protein. A fusion protein may also be engineered tocontain a cleavage site located between a PHIT sequence and the non-FHITprotein sequence, so that the FHIT protein may be cleaved away from thenon-FHIT moiety. In a specific embodiment, the FHIT amino acid sequencepresent in the fusion protein consists of at least 10 contiguous aminoacids, at least 25 contiguous amino acids, at least 50 contiguous aminoacids, at least 75 contiguous amino acids, at least 100 contiguous aminoacids, or at least 147 amino acids (full-length) of the Fhit proteinsequence.

In an alternate embodiment of the invention, the coding sequence of aFHIT is synthesized in whole or in part, using chemical methods wellknown in the art. See, for example, Caruthers et al., 1980, Nuc. AcidsRes. Symp. Ser. 7:215-233; Crea and Horn, 1980, Nuc. Acids Res.9(10):2331; Matteucci and Caruthers, 1980, Tetrahedron Letters 21:719;and Chow and Kempe, 1981, Nuc. Acids Res. 9(12):2807-2817.Alternatively, the protein itself could be produced using chemicalmethods to synthesize an FHIT amino acid sequence in whole or in part.For example, peptides can be synthesized by solid phase techniques,cleaved from the resin, and purified by preparative high performanceliquid chromatography. (e.g., see Creighton, 1983, Proteins StructuresAnd Molecular Principles, W.H. Freeman and Co., N.Y. pp. 50-60). Thecomposition of the synthetic peptides may be confirmed by amino acidanalysis or sequencing (e.g., the Edman degradation procedure; seeCreighton, 1983, Proteins, Structures and Molecular Principles, W.H.Freeman and Co., N.Y., pp. 34-49.

In order to express a biologically active FHIT protein or derivativethereof, a polynucleotide sequence encoding a FHIT protein, or aderivative thereof, is inserted into an appropriate-expression vector,i.e., a vector which contains the necessary elements for thetranscription and translation of the inserted coding sequence. The FHITgene products as well as host cells or cell lines transfected ortransformed with recombinant FHIT expression vectors can be used for avariety of purposes. These include but are not limited to generatingantibodies (i.e., monoclonal or polyclonal) that immunospecifically binda FHIT protein. Anti-FHIT antibodies can be used in detecting ormeasuring levels of a FHIT protein in patient samples.

5.2.1. Expression Systems

Methods which are well known to those skilled in the art can be used toconstruct expression vectors containing a FHIT coding sequence andappropriate transcriptional/translational control signals. These methodsinclude in vitro recombinant DNA techniques, synthetic techniques and invivo recombination/genetic recombination. See, for example, thetechniques described in Sambrook et al., 1989, Molecular Cloning, ALaboratory Manual 2d ed., Cold Spring Harbor Laboratory, N.Y. andAusubel et al., 1989, Current Protocols in Molecular Biology, GreenePublishing Associates and Wiley Interscience, N.Y.

A variety of host-expression vector systems may be utilized to express aFHIT coding sequence. These include but are not limited tomicroorganisms such as bacteria transformed with recombinantbacteriophage DNA, plasmid DNA or cosmid DNA expression vectorscontaining an PHIT coding sequence; yeast transformed with recombinantyeast expression vectors containing an FHIT coding sequence; insect cellsystems infected with recombinant virus expression vectors (e.g.,baculovirus) containing an PHIT coding sequence; plant cell systemsinfected with recombinant virus expression vectors (e.g., cauliflowermosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed withrecombinant plasmid expression vectors (e.g., Ti plasmid) containing anFHIT coding sequence; or animal cell systems. The expression elements ofthese systems vary in their strength and specificities. Depending on thehost/vector system utilized, any of a number of suitable transcriptionand translation elements, including constitutive and induciblepromoters, may be used in the expression vector. For example, whencloning in bacterial systems, inducible promoters such as pL ofbacteriophage λ, plac, ptrp, ptac (ptrp-lac hybrid promoter) and thelike may be used; when cloning in insect cell systems, promoters such asthe baculovirus polyhedrin promoter may be used; when cloning in plantcell systems, promoters derived from the genome of plant cells (e.g.,heat shock promoters; the promoter for the small subunit of RUBISCO; thepromoter for the chlorophyll a/b binding protein) or from plant viruses(e.g., the 35S RNA promoter of CaMV; the coat protein promoter of TMV)may be used; when cloning in mammalian cell systems, promoters derivedfrom the genome of mammalian cells (e.g., metallothionein promoter) orfrom mammalian viruses (e.g., the adenovirus late promoter; the vacciniavirus 7.5 K promoter) may be used; when generating cell lines thatcontain multiple copies of an FHIT DNA, SV40-, BPV- and EBV-basedvectors may be used with an appropriate selectable marker.

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the FHITprotein expressed. For example, when large quantities of FHIT proteinare to be produced for the generation of antibodies, vectors whichdirect the expression of high levels of fusion protein products that arereadily purified may be desirable. Such vectors include but are notlimited to the E. coli expression vector pUR278 (Ruther et al., 1983,EMBO J. 2:1791), in which the FHIT coding sequence may be ligated intothe vector in frame with the lac Z coding region so that a hybrid AS-lacZ protein is produced; pIN vectors (Inouye & Inouye, 1985, Nucleic acidsRes. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem.264:5503-5509); and the like. pGEX vectors may also be used to expressforeign polypeptides as fusion proteins with glutathione S-transferase(GST) (Smith and Johnson, 1988, Gene 7:31-40). In general, such fusionproteins are soluble and can easily be purified from lysed cells byadsorption to glutathione-agarose beads followed by elution in thepresence of free glutathione. The pGEX vectors are designed to includethrombin or factor Xa protease cleavage sites so that the clonedpolypeptide of interest can be released from the GST moiety.

In yeast, a number of vectors containing constitutive or induciblepromoters may be used. For a review see, Current Protocols in MolecularBiology, Vol. 2, 1988, Ed. Ausubel et al., Greene Publish. Assoc. &Wiley Interscience, Ch. 13; Grant et al., 1987, Expression and SecretionVectors for Yeast, in Methods in Enzymology, Ed. Wu & Grossman, 1987,Acad. Press, N.Y. 153:516-544; Glover, 1986, DNA Cloning, Vol. II, IRLPress, Wash., D.C., Ch. 3; and Bitter, 1987, Heterologous GeneExpression in Yeast, Methods in Enzymology, Eds. Berger & Kimmel, Acad.Press, N.Y. 152:673-684; and The Molecular Biology of the YeastSaccharomyces, 1982, Eds. Strathern et al., Cold Spring. Harbor Press,Vols. I and II.

In cases where plant expression vectors are used, the expression of anFHIT coding sequence may be driven by any of a number of promoters. Forexample, viral promoters such as the 35S RNA and 19S RNA promoters ofCaMV (Brisson et al., 1984, Nature 310:511-514), or the coat proteinpromoter of TMV (Takamatsu et al., 1987, EMBO J. 6:307-311) may be used;alternatively, plant promoters such as the small subunit of RUBISCO(Coruzzi et al., 1984, EMBO J. 3:1671-1680; Broglie et al., 1984,Science 224:838-843); or heat shock promoters, e.g., soybean hsp17.5-Eor hsp17.3-B (Gurley et al., 1986, Mol. Cell. Biol. 6:559-565) may beused. These constructs can be introduced into plant cells using Tiplasmids, Ri plasmids, plant virus vectors, direct DNA transformation,microinjection, electroporation, etc. For reviews of such techniquessee, for example, Weissbach & Weissbach, 1988, Methods for PlantMolecular Biology, Academic Press, NY, Section VIII, pp. 421-463; andGrierson & Corey, 1988, Plant Molecular Biology, 2d Ed., Blackie,London, Ch. 7-9.

An alternative expression system which could be used to express a FHITgene is an insect system. In one such system, Autographa californicanuclear polyhedrosis virus (AcNPV) is used as a vector to expressforeign genes. The virus grows in Spodoptera frugiperda cells. A FHITcoding sequence may be cloned into non-essential regions (for examplethe polyhedrin gene) of the virus and placed under control of an AcNPVpromoter (for example, the polyhedrin promoter). Successful insertion ofa FHIT coding sequence will result in inactivation of the polyhedringene and production of non-occluded recombinant virus (i.e., viruslacking the proteinaceous coat coded for by the polyhedrin gene). Theserecombinant viruses are then used to infect Spodoptera frugiperda cellsin which the inserted gene is expressed. (e.g., see Smith et al., 1983,J. Virol. 46:584; Smith, U.S. Pat. No. 4,215,051).

In mammalian host cells, a number of viral based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, a FHIT coding sequence may be ligated to an adenovirustranscription/translation control complex, e.g., the late promoter andtripartite leader sequence. This chimeric gene may then be inserted inthe adenovirus genome by in vitro or in vivo recombination. Insertion ina non-essential region of the viral genome (e.g., region E1 or E3) willresult in a recombinant virus that is viable and capable of expressing aFHIT in infected hosts. (e.g., see Logan & Shenk, 1984, Proc. Natl.Acad. Sci. USA 81:3655-3659). Alternatively, the vaccinia 7.5 K promotermay be used. (See, e.g., Hackett et al., 1982, Proc. Natl. Acad. Sci.USA 79:7415-7419; Madkett et al., 1984, J. Virol. 49:857-864; Panicaliet al., 1982, Proc. Natl. Acad. Sci. USA 79:4927-4931).

Specific initiation signals may also be required for efficienttranslation of an inserted FHIT coding sequences. These signals includethe ATG initiation codon and adjacent sequences. In cases where anentire FRIT gene, including its own initiation codon and adjacentsequences, is inserted into the appropriate expression vector, noadditional translational control signals may be needed. However, incases where only a portion of a FHIT coding sequence is inserted,lacking the 5′ end, exogenous translational control signals, includingthe ATG initiation codon, must be provided. Furthermore, the initiationcodon must be in phase with the reading frame of a FHIT coding sequenceto ensure translation of the entire insert. These exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression maybe enhanced by the inclusion of appropriate transcription enhancerelements, transcription terminators, etc. (see Bittner et al., 1987,Methods in Enzymol. 153:516-544).

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,phosphorylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins. Appropriate cells lines or hostsystems can be chosen to ensure the correct modification and processingof the foreign protein expressed. To this end, eukaryotic host cellswhich possess the cellular machinery for proper processing of theprimary transcript, and phosphorylation of the gene product may be used.Such mammalian host cells include but are not limited to CHO, VERO, BHK,HeLa, COS, MDCK, 293, WI38, etc.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably express aFHIT protein may be engineered. Rather than using expression vectorswhich contain viral origins of replication, host cells can betransformed with FHIT DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express a FHITprotein. The present invention provides a method for producing arecombinant FHIT protein comprising culturing a host cell transformedwith a recombinant expression vector encoding a FHIT protein such thatthe FHIT protein is expressed by the cell and recovering the expressedFHIT protein.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), and adeninephosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes can beemployed in tk−, hgprt− or aprt− cells, respectively. Also,antimetabolite resistance can be used as the basis of selection fordhfr, which confers resistance to methotrexate (Wigler et al., 1980,Natl. Acad. Sci. USA 77:3567; O'Hare et al., 1981, Proc. Natl. Acad.Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid(Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, whichconfers resistance to the aminoglycoside G-418 (Colberre-Garapin et al.,1981, J. Mol. Biol. 150:1); and hygro, which confers resistance tohygromycin (Santerre et al., 1984, Gene 30:147). Recently, additionalselectable genes have been described, namely trpB, which allows cells toutilize indole in place of tryptophan; hisD, which allows cells toutilize histinol in place of histidine (Hartman & Mulligan, 1988, Proc.Natl. Acad. Sci. USA 85:8047); and ODC (ornithine decarboxylase) whichconfers resistance to the ornithine decarboxylase inhibitor,2-(difluoromethyl)-DL-ornithine, DFMO (McConlogue, L., 1987, In: CurrentCommunications in Molecular Biology, Cold Spring Harbor Laboratory,Ed.).

5.2.2. Identification of Transfectants or Transformants That ExpressFHIT

The host cells which contain the coding sequence and which express thebiologically active gene product may be identified by at least fourgeneral approaches; (a) DNA-DNA or DNA-RNA hybridization; (b) thepresence or absence of “marker” gene functions; (c) assessing the levelof transcription as measured by the expression of FHIT mRNA transcriptsin the host cell; and (d) detection of the gene product as measured byimmunoassay or by its biological activity.

In the first approach, the presence of the FHIT coding sequence insertedin the expression vector can be detected by DNA-DNA or DNA-RNAhybridization using probes comprising nucleotide sequences that arehomologous to the FHIT coding sequence, respectively, or portions orderivatives thereof.

In the second approach, the recombinant expression vector/host systemcan be identified and selected based upon the presence or absence ofcertain “marker” gene functions (e.g., thymidine kinase activity,resistance to antibiotics, resistance to methotrexate, transformationphenotype, occlusion body formation in baculovirus, etc.). For example,if the human FHIT coding sequence is inserted within a marker genesequence of the vector, recombinant cells containing the FHIT codingsequence can be identified by the absence of the marker gene function.Alternatively, a marker gene can be placed in tandem with a FHITsequence under the control of the same or different promoter used tocontrol the expression of the FHIT coding sequence. Expression of themarker in response to induction or selection indicates expression of theFHIT coding sequence.

In the third approach, transcriptional activity of a FHIT gene can beassessed by hybridization assays. For example, RNA can be isolated andanalyzed by Northern-blot using a probe having sequence homology to aFHIT coding sequence or transcribed noncoding sequence or particularportions thereof. Alternatively, total nucleic acid of the host cell maybe extracted and quantitatively assayed for hybridization to suchprobes.

In the fourth approach, the levels of a FHIT protein product can beassessed immunologically, for example by Western blots, immunoassayssuch as radioimmuno-precipitation, enzyme-linked immunoassays and thelike.

5.3. Purification of the Expressed Gene Product

Once a recombinant which expresses the FHIT gene sequence is identified,the gene product can be analyzed. This is achieved by assays based onthe physical or functional properties of the product, includingradioactive labelling of the product followed by analysis by gelelectrophoresis, immunoassay, or other detection methods known to thoseof skill in the art.

Once the FHIT protein is identified, it may be isolated and purified bystandard methods including chromatography (e.g., ion exchange, affinity,and sizing column chromatography), centrifugation, differentialsolubility, or by any other standard technique for the purification ofproteins. The functional properties may be evaluated using any suitableassay.

Alternatively, once a FHIT protein produced by a recombinant isidentified, the amino acid sequence of the protein can be deduced fromthe nucleotide sequence of the chimeric gene contained in therecombinant. As a result, the protein can be synthesized by standardchemical methods known in the art (e.g., see Hunkapiller et al., 19.84,Nature 310:105-111).

In a specific embodiment of the present invention, such FHIT proteins,whether produced by recombinant DNA techniques or by chemical syntheticmethods, include but are not limited to those containing, as a primaryamino acid sequence, all or part of the amino acid sequencesubstantially as depicted in FIG. 2A (SEQ ID NO:2), as well as fragmentsand other derivatives, and analogs thereof.

5.4. Generation of Antibodies to Fhit

According to the invention, Fhit protein, its fragments or otherderivatives, or analogs thereof, may be used as an immunogen to generateantibodies which recognize such an immunogen. Such antibodies includebut are not limited to polyclonal, monoclonal, chimeric, single chain,Fab fragments, and an Fab expression library. In a specific embodiment,antibodies to a human Fhit protein are produced.

Various procedures known in the art may be used for the production ofpolyclonal antibodies to a Fhit protein or derivative or analog. For theproduction of antibody, various host animals can be immunized byinjection with the native Fhit protein, or a synthetic version, orderivative (e.g., fragment) thereof, including but not limited torabbits, mice, rats, etc. Various adjuvants may be used to increase theimmunological response, depending on the host species, and including butnot limited to Freund's (complete and incomplete), mineral gels such asaluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanins, dinitrophenol, and potentially useful human adjuvants suchas BCG (bacille Calmette-Guerin) and corynebacterium parvum.

For preparation of monoclonal antibodies directed toward a Fhit proteinsequence or analog thereof, any technique which provides for theproduction of antibody molecules by continuous cell lines in culture maybe used. For example, the hybridoma technique originally developed byKohler and Milstein (1975, Nature 256:495-497), as well as the triomatechnique, the human B-cell hybridoma technique (Kozbor et al., 1983,Immunology Today 4:72), and the EBV-hybridoma technique to produce humanmonoclonal antibodies (Cole et al., 1985, in Monoclonal Antibodies andCancer Therapy, Alan R. Liss, Inc., pp. 77-96). In an additionalembodiment of the invention, monoclonal antibodies can be produced ingerm-free animals utilizing recent technology (PCT/US90/02545).According to the invention, human antibodies may be used and can beobtained by using human hybridomas (Cote et al., 1983, Proc. Natl. Acad.Sci. USA 80:2026-2030) or by transforming human B cells with EBV virusin vitro (Cole et al., 1985, in Monoclonal Antibodies and CancerTherapy, Alan R. Liss, pp. 77-96). In fact, according to the invention,techniques developed for the production of “chimeric antibodies”(Morrison et al., 1984, Proc. Natl. Acad. Sci. USA 81:6851-6855;Neuberger et al., 1984, Nature 312:604-608; Takeda et al., 1985, Nature314:452-454) by splicing the genes from a mouse antibody moleculespecific for FHIT together with genes from a human antibody molecule ofappropriate biological activity can be used; such antibodies are withinthe scope of this invention.

According to the invention, techniques described for the production ofsingle chain antibodies (U.S. Pat. No. 4,946,778) can be adapted toproduce Fhit-specific single chain antibodies. An additional embodimentof the invention utilizes the techniques described for the constructionof Fab expression libraries (Huse et al., 1989, Science 246:1275-1281)to allow rapid and easy identification of monoclonal Fab fragments withthe desired specificity for Fhit proteins, derivatives, or analogs.

In a specific embodiment, a molecule comprising a fragment of the Fhitprotein is used as an immunogen. For example, since hydrophilic regionsare believed most likely to contain antigenic determinants, a peptidecorresponding to or containing a hydrophilic portion of a Fhit proteinis preferably used as immunogen.

Antibody fragments which contain the idiotype of the molecule can begenerated by known techniques. For example, such fragments include butare not limited to: the F(ab′)₂ fragment which can be produced by pepsindigestion of the antibody molecule; the Fab′ fragments which can begenerated by reducing the disulfide bridges of the F(ab′)₂ fragment, andthe Fab fragments which can be generated by treating the antibodymolecule with papain and a reducing agent.

In the production of antibodies, screening for the desired antibody canbe accomplished by techniques known in the art, e.g. ELISA(enzyme-linked immunosorbent assay). For example, to select antibodieswhich recognize a specific domain of a Fhit protein, one may assaygenerated hybridomas for a product which binds to a Fhit fragmentcontaining such domain. For selection of an antibody specific to humanFhit, one can select on the basis of positive binding to human Fhit anda lack of binding to, for example, mouse Fhit.

The foregoing antibodies can be used in methods known in the artrelating to the localization and activity of the protein sequences ofthe invention, e.g., for imaging these proteins, measuring levelsthereof in appropriate physiological samples, in diagnostic methods,etc.

5.5. Structure of the FHIT Gene and Protein

The structure of the FHIT gene and protein can be analyzed by variousmethods known in the art.

5.5.1. Genetic Analysis

The cloned DNA or cDNA-corresponding to the FHIT gene can be analyzed bymethods including but not limited to Southern hybridization (Southern,E. M., 1975, J. Mol. Biol. 98:503-517), Northern hybridization (see,e.g., Freeman et al., 1983, Proc. Natl. Acad. Sci. USA 80:4094-4098),restriction endonuclease mapping (Maniatis, T., 1982, Molecular Cloning,A Laboratory, Cold Spring Harbor, N.Y.), and DNA sequence analysis.Polymerase chain reaction (PCR; U.S. Pat. Nos. 4,683,202, 4,683,195, and4,889,818; Gyllenstein et al., 1988, Proc. Natl. Acad. Sci. USA85:7652-7656; Ochman et al., 1988, Genetics 120:621-623; Loh et al.,1989, Science 243:217-220) followed by Southern hybridization with aFHIT-specific probe can allow the detection of the FHIT gene in DNA fromvarious cell types. In one embodiment, Southern hybridization may beused to determine the genetic linkage of FHIT. PCR followed byhybridization assay can also be used to detect or measure FHIT RNA or3p14.2 chromosomal or molecular abnormalities. Northern hybridizationanalysis can be used to determine the expression levels of the PHITgene. Other assays are described in Section 5.11. Various cell types, atvarious states of development or activity can be tested for FHITexpression. The stringency of the hybridization conditions for bothSouthern and Northern hybridization, or dot blots, can be manipulated toensure detection of nucleic acids with the desired degree of relatednessto the specific FHIT probe used.

Restriction endonuclease mapping can be used to roughly determine thegenetic structure of the FHIT gene. Restriction maps derived byrestriction endonuclease cleavage can be confirmed by DNA sequenceanalysis.

DNA sequence analysis can be performed by any techniques known in theart, including but not limited to the method of Maxam and Gilbert (1980,Meth. Enzymol. 65:499-560), the Sanger dideoxy method (Sanger et al.,1977, Proc. Natl. Acad. Sci. USA 74:5463), the use of T7 DNA polymerase(Tabor and Richardson, U.S. Pat. No. 4,795,699), or use of an automatedDNA sequenator (e.g., Applied Biosystems, Foster City, Calif.). The cDNAsequence of a representative FHIT gene comprises the sequencesubstantially as depicted in FIG. 2A (SEQ ID NO: 1), and described inSection 6, infra.

5.5.2. Protein Analysis

The amino acid sequence of the Fhit protein can be derived by deductionfrom the DNA sequence, or alternatively, by direct sequencing of theprotein, e.g., with an automated amino acid sequencer. The amino acidsequence of a representative Fhit protein comprises the sequencesubstantially as depicted in FIG. 2A (SEQ ID NO: 2), and detailed inSection 6, infra, with the representative mature protein that is shownby amino acid numbers 1-147.

The Fhit protein sequence can be further characterized by ahydrophilicity analysis (Hopp, T. and Woods, K., 1981, Proc. Natl. Acad.Sci. USA 78:3824). A hydrophilicity profile can be used to identify thehydrophobic and hydrophilic regions of the Fhit protein and thecorresponding regions of the gene sequence which encode such regions.

Secondary structural analysis (Chou, P. and Fasman, G., 1974,Biochemistry 13:222) can also be done, to identify regions of the Fhitprotein that assume specific secondary structures.

Manipulation, translation, and secondary structure prediction, as wellas open reading frame prediction and plotting, can also be accomplishedusing computer software programs available in the art.

Other methods of structural analysis can also be employed. These includebut are not limited to X-ray crystallography (Engstom, A., 1974,Biochem. Exp. Biol. 11:7-13) and computer modeling (Fletterick, R. andZoller, M. (eds.), 1986, Computer Graphics and Molecular Modeling, inCurrent Communications in Molecular Biology, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.).

5.6. Fhit Proteins, Derivatives and Analogs

The invention further relates to Fhit proteins, and derivatives(including but not limited to fragments) and analogs of Fhit proteins.Nucleic acids encoding Fhit protein derivatives and protein analogs arealso provided. Molecules comprising Fhit proteins or derivatives arealso provided. In one embodiment, the Fhit proteins are encoded by theFhit nucleic acids described in Section 5.1 supra. In particularaspects, the proteins, derivatives, or analogs are of Fhit proteins ofanimals.

The production and use of derivatives and analogs related to Fhit arewithin the scope of the present invention. In a specific embodiment, thederivative or analog is functionally active, i.e., capable of exhibitingone or more functional activities associated with a full-length,wild-type Fhit protein. As one example, such derivatives or analogswhich have the desired immunogenicity or antigenicity can be used, forexample, in immunoassays, for immunization, for inhibition of Fhitactivity, etc. As another example, such derivatives or analogs whichhave hydrolase activity are provided. Derivatives or analogs thatretain, or alternatively lack or inhibit, a desired Fhit property ofinterest (e.g., inhibition of cell proliferation, tumor inhibition), canbe used as inducers, or inhibitors, respectively, of such property andits physiological correlates. A specific embodiment relates to a Fhitfragment that can be bound by an anti-Fhit antibody. Derivatives oranalogs of Fhit can be tested for the desired activity by proceduresknown in the art, including but not limited to the assays describedinfra.

In particular, Fhit derivatives can be made by altering FHIT sequencesby substitutions, additions or deletions that provide for functionallyequivalent molecules. Due to the degeneracy of nucleotide codingsequences, other DNA sequences which encode substantially the same aminoacid sequence as a FHIT gene may be used in the practice of the presentinvention. These include but are not limited to nucleotide sequencescomprising all or portions of FHIT genes which are altered by thesubstitution of different codons that encode a functionally equivalentamino acid residue within the sequence, thus producing a silent change.Likewise, the Fhit derivatives of the invention include, but are notlimited to, those containing, as a primary amino acid sequence, all orpart of the amino acid sequence of a Fhit protein including alteredsequences in which functionally equivalent amino acid residues aresubstituted for residues within the sequence resulting in a silentchange. For example, one or more amino acid residues within the sequencecan be substituted by another amino acid of a similar polarity whichacts as a functional equivalent, resulting in a silent alteration.Substitutes for an amino acid within the sequence may be selected fromother members of the class to which the amino acid belongs. For example,the nonpolar (hydrophobic) amino acids include alanine, leucine,isoleucine, valine, proline, phenylalanine, tryptophan and methionine.The polar neutral amino acids include glycine, serine, threonine,cysteine, tyrosine, asparagine, and glutamine. The positively charged(basic) amino acids include arginine, lysine and histidine. Thenegatively charged (acidic) amino acids include aspartic acid andglutamic acid.

In a specific embodiment of the invention, proteins consisting of orcomprising a fragment of a Fhit protein consisting of at least 10(continuous) amino acids of the Fhit protein is provided. In otherembodiments, the fragment consists of at least 20 or 50 amino acids ofthe Fhit protein. In specific embodiments, such fragments are not largerthan 35, 100 or 140 amino acids. Derivatives or analogs of Fhit includebut are not limited to those molecules comprising regions that aresubstantially homologous to Fhit or fragments thereof (e.g., in variousembodiments, at least 60% or 70% or 80% or 90% or 95% identity over anamino acid sequence of identical size or when compared to an alignedsequence in which the alignment is done by a computer homology programknown in the art) or whose encoding nucleic acid is capable ofhybridizing to a coding FHIT sequence, under stringent, moderatelystringent, or nonstringent conditions.

The Fhit derivatives and analogs of the invention can be produced byvarious methods known in the art. The manipulations which result intheir production can occur at the gene or protein level. For example,the cloned FHIT gene sequence can be modified by any of numerousstrategies known in the art (Maniatis, T., 1990, Molecular Cloning, ALaboratory Manual, 2d ed., Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.). The sequence can be cleaved at appropriate sites withrestriction endonuclease(s), followed by further enzymatic modificationif desired, isolated, and ligated in vitro. In the production of thegene encoding a derivative or analog of Fhit, care should be taken toensure that the modified gene remains within the same translationalreading frame as Fhit, uninterrupted by translational stop signals, inthe gene region where the desired Fhit activity is encoded.

Additionally, the Fhit-encoding nucleic acid sequence can be mutated invitro or in vivo, to create and/or destroy translation, initiation,and/or termination sequences, or to create variations in coding regionsand/or form new restriction endonuclease sites or destroy preexistingones, to facilitate further in vitro modification. Any technique formutagenesis known in the art can be used, including but not limited to,chemical mutagenesis, in vitro site-directed mutagenesis (Hutchinson,C., et al., 1978, J. Biol. Chem 253:6551), etc.

Manipulations of the Fhit sequence may also be made at the proteinlevel. Included within the scope of the invention are Fhit proteinfragments or other derivatives or analogs which are differentiallymodified during or after translation, e.g., by glycosylation,acetylation, phosphorylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, linkage to an antibodymolecule or other cellular ligand, etc. Any of numerous chemicalmodifications may be carried out by known techniques, including but notlimited to specific chemical cleavage by cyanogen bromide, trypsin,chymotrypsin, papain, V8 protease, NaBH₄; acetylation, formylation,oxidation, reduction; metabolic synthesis in the presence oftunicamycin; etc.

In addition, analogs and derivatives of Fhit can be chemicallysynthesized. For example, a peptide corresponding to a portion of a Fhitprotein which comprises the desired domain (see Section 5.6.1), or whichmediates the desired activity in vitro, can be synthesized by use of apeptide synthesizer. Furthermore, if desired, nonclassical amino acidsor chemical amino acid analogs can be introduced as a substitution oraddition into the Fhit sequence. Non-classical amino acids include butare not limited to the D-isomers of the common amino acids, α-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, γ-Abu,ε-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline,sarcosine, citrulline, cysteic acid, t-butylglycine, t-butylalanine,phenylglycine, cyclohexylalanine, β-alanine, fluoro-amino acids,designer amino acids such as β-methyl amino acids, Cα-methyl aminoacids, Nα-methyl amino acids, and amino acid analogs in general.Furthermore, the amino acid can be D (dextrorotary) or L (levorotary).

In a specific embodiment, the Fhit derivative is a chimeric, or fusion,protein comprising a Fhit protein or fragment thereof (preferablyconsisting of at least a domain or motif of the Fhit protein, or atleast 10 amino acids of the Fhit protein) joined at its amino- orcarboxy-terminus via a peptide bond to an amino acid sequence of adifferent protein. In one embodiment, such a chimeric protein isproduced by recombinant expression of a nucleic acid encoding theprotein (comprising a Fhit-coding sequence joined in-frame to a codingsequence for a different protein). Such a chimeric product can be madeby ligating the appropriate nucleic acid sequences encoding the desiredamino acid sequences to each other by methods known in the art, in theproper coding frame, and expressing the chimeric product by methodscommonly known in the art. Alternatively, such a chimeric product may bemade by protein synthetic techniques, e.g., by use of a peptidesynthesizer. Chimeric genes comprising portions of FHIT fused to anyheterologous protein-encoding sequences may be constructed.

In another specific embodiment, the Fhit derivative is a moleculecomprising a region of homology with a Fhit protein. By way of example,in various embodiments, a first protein region can be considered“homologous” to a second protein region when the amino acid sequence ofthe first region is at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, or95% identical, when compared to any sequence in the second region of anequal number of amino acids as the number contained in the first regionor when compared to an aligned sequence of the second region that hasbeen aligned by a computer homology program known in the art. Forexample, a molecule can comprise one or more regions homologous to aFhit domain (see Section 5.6.1) or a portion thereof or a full-lengthFhit protein.

5.7. Assays of Fhit Proteins, Derivatives and Analogs

The functional activity of Fhit proteins, derivatives and analogs can beassayed by various methods.

For example, in one embodiment, where one is assaying for the ability tobind or compete with wild-type Fhit for binding to anti-Fhit antibody,various immunoassays known in the art can be used, including but notlimited to competitive and non-competitive assay systems usingtechniques such as radioimmunoassays, ELISA (enzyme linked immunosorbentassay), “sandwich” immunoassays, immunoradiometric assays, gel diffusionprecipitin reactions, immunodiffusion assays, in situ immunoassays(using colloidal gold, enzyme or radioisotope labels, for example),western blots, precipitation reactions, agglutination assays (e.g., gelagglutination assays, hemagglutination assays), complement fixationassays, immunofluorescence assays, protein A assays, andimmunoelectrophoresis assays, etc. In one embodiment, antibody bindingis detected by detecting a label on the primary antibody. In anotherembodiment, the primary antibody is detected by detecting binding of asecondary antibody or reagent to the primary antibody. In a furtherembodiment, the secondary antibody is labelled. Many means are known inthe art for detecting binding in an immunoassay and are within the scopeof the present invention.

In another embodiment, where a Fhit-binding protein is identified, thebinding can be assayed, e.g., by means well-known in the art.

In another embodiment, should a Fhit protein have hydrolase activity,hydrolase assays can be used to measure Fhit hydrolase activity. Suchassays can be carried out by methods well known in the art.

In addition, assays known in the art can be used to detect or measurethe ability to inhibit cell proliferation, in vitro or in vivo.

Other methods will be known to the skilled artisan and are within thescope of the invention.

5.8. Therapeutic Uses: Treatment and Prevention of Disorders InvolvingOverproliferation of Cells

The invention provides for treatment or prevention of various diseasesand disorders by administration of a therapeutic compound (termed herein“Therapeutic”). Such “Therapeutics” include but are not limited to: Fhitproteins and analogs and derivatives (including fragments) thereof(e.g., as described hereinabove); antibodies thereto (as describedhereinabove); nucleic acids encoding the Fhit proteins, analogs, orderivatives (e.g., as described hereinabove); and Fhit agonists, andantagonists of mutant FHIT genes or proteins (e.g., antibodies orantisense nucleic acids). In a preferred embodiment, disorders involvingcell overproliferation are treated or prevented by administration of aTherapeutic that promotes Fhit function. The above is described indetail in the subsections below.

Generally, administration of products of a species origin or speciesreactivity (in the case of antibodies) that is the same species as thatof the patient is preferred. Thus, in a preferred embodiment, a humanFhit protein, derivative, or analog, or nucleic acid, or an antibody toa human Fhit protein or human FHIT antisense nucleic acid, istherapeutically or prophylactically administered to a human patient.

Additional descriptions and sources of Therapeutics that can be usedaccording to the invention are found in Sections 5.1 through 5.7 herein.

A FHIT polynucleotide and its Fhit protein product can be used fortherapeutic/prophylactic purposes for diseases involving celloverproliferation, as well as other disorders associated withchromosomal translocations or inversions or molecular abnormalitiesassociated with the FHIT locus, and/or decreased expression of wild-typeFHIT RNA or protein and/or expression of a mutant FHIT RNA or proteinand/or expression of a mutant FHIT RNA or protein. A FHITpolynucleotide, and its FHIT protein product, may be used fortherapeutic/prophylactic purposes alone or in combination with othertherapeutics useful in the treatment of cancer and hyperproliferative ordysproliferative disorders.

Diseases and disorders involving cell overproliferation are treated orprevented by administration of a Therapeutic that promotes (i.e.,increases or supplies) Fhit function. Examples of such a Therapeuticinclude but are not limited to Fhit proteins, derivatives, or fragmentsthat are functionally active, particularly that are active in inhibitingcell proliferation (e.g., as demonstrated in in vitro assays or inanimal models), and nucleic acids encoding a Fhit protein orfunctionally active derivative or fragment thereof (e.g., for use ingene therapy). Other Therapeutics that can be used, e.g., Fhit agonists,can be identified using in vitro assays or animal models, examples ofwhich are described infra.

In specific embodiments, Therapeutics that promote Fhit function areadministered therapeutically (including prophylactically): (1) indiseases or disorders involving an absence or decreased (relative tonormal or desired) level of Fhit functional protein or of Fhit function,for example, in patients where Fhit protein is lacking, geneticallydefective, biologically inactive or underactive, or underexpressed; or(2) in diseases or disorders wherein in vitro (or in vivo) assaysindicate the utility of Fhit agonist administration. The absence ordecreased level in Fhit protein or function can be readily detected,e.g., by obtaining a patient tissue sample (e.g., from biopsy tissue)and assaying it in vitro for RNA or protein levels, structure and/oractivity of the expressed Fhit RNA or protein (see Section 5.11 infra reassays used in diagnosis). Many methods standard in the art can be thusemployed, including but not limited to immunoassays to detect and/orvisualize Fhit protein (e.g., Western blot, immunoprecipitation followedby sodium dodecyl sulfate polyacrylamide gel electrophoresis,immunocytochemistry, etc.) and/or hybridization assays to detect Fhitexpression by detecting and/or visualizing Fhit mRNA or cDNA (e.g.,Northern assays, dot blots, in situ hybridization, and preferably thoseassays described in Section 5.11), etc.

Diseases and disorders involving cell overproliferation that can betreated or prevented include but are not limited to malignancies,premalignant conditions (e.g., hyperplasia, metaplasia, dysplasia),benign tumors, hyperproliferative disorders, benign dysproliferativedisorders, etc. Examples of these are detailed below.

5.8.1. Malignancies

Malignancies and related disorders that can be treated or prevented byadministration of a Therapeutic that promotes Fhit function (e.g., afull-length Fhit protein or functional derivative thereof or nucleicacid encoding the foregoing) include but are not limited to those listedin Table 1 (for a review of such disorders, see Fishman et al., 1985,Medicine, 2d Ed., J.B. Lippincott Co., Philadelphia): TABLE 1MALIGNANCIES AND RELATED DISORDERS Leukemia   acute leukemia     acutelymphocytic leukemia     acute myelocytic leukemia       myeloblastic      promyelocytic       myelomonocytic       monocytic      erythroleukemia   chronic leukemia     chronic myelocytic(granulocytic) leukemia     chronic lymphocytic leukemia    Polycythemia vera Lymphoma   Hodgkin's disease   non-Hodgkin'sdisease Multiple myeloma Waldenström's macroglobulinemia Heavy chaindisease Solid tumors   sarcomas and carcinomas     fibrosarcoma    myxosarcoma     liposarcoma     chondrosarcoma     osteogenicsarcoma     osteosarcoma     chordoma     angiosarcoma    endotheliosarcoma     lymphangiosarcoma    lymphangioendotheliosarcoma     synovioma     mesothelioma    Ewing's tumor     leiomyosarcoma     rhabdomyosarcoma     coloncarcinoma     colorectal carcinoma     pancreatic cancer     breastcancer     ovarian cancer     prostate cancer     squamous cellcarcinoma     basal cell carcinoma     adenocarcinoma     sweat glandcarcinoma     sebaceous gland carcinoma     papillary carcinoma    papillary adenocarcinomas     cystadenocarcinoma     medullarycarcinoma     bronchogenic carcinoma     renal cell carcinoma    hepatoma     bile duct carcinoma     choriocarcinoma     seminoma    embryonal carcinoma     Wilms' tumor     cervical cancer     uterinecancer     testicular tumor     lung carcinoma     small cell lungcarcinoma     non small cell lung carcinoma     bladder carcinoma    epithelial carcinoma     glioma     astrocytoma     medulloblastoma    craniopharyngioma     ependymoma     pinealoma     hemangioblastoma    acoustic neuroma     oligodendroglioma     menangioma     melanoma    neuroblastoma     retinoblastoma     nasopharyngeal carcinoma    esophageal carcinoma

In a specific embodiment, digestive tract tumors are treated orprevented, including but not limited to esophageal, stomach, colon, andcolorectal cancers. In another specific embodiment, airway cancers suchas lung cancers (e.g., small cell lung carcinoma) and nasopharyngealcarcinoma are treated or prevented. In yet other specific embodiments,malignancy or dysproliferative changes (such as metaplasias anddysplasias), or hyperproliferative disorders, are treated or preventedin the head, neck, cervix, kidney, stomach, skin, ovary, bladder,breast, colon, lung, or uterus. In other specific embodiments, sarcoma,or leukemia is treated or prevented. In another particular embodiment,osteosarcoma or renal cell carcinoma is treated or prevented.

5.8.2. Premalignant Conditions

The Therapeutics of the invention that promote Fhit activity can also beadministered to treat premalignant conditions and to prevent progressionto a neoplastic or malignant state, including but not limited to thosedisorders listed in Table 1. Such prophylactic or therapeutic use isindicated in conditions known or suspected of preceding progression toneoplasia or cancer, in particular, where non-neoplastic cell growthconsisting of hyperplasia, metaplasia, or most particularly, dysplasiahas occurred (for review of such abnormal growth conditions, see Robbinsand Angell, 1976, Basic Pathology, 2d Ed., W.B. Saunders Co.,Philadelphia, pp. 68-79.) Hyperplasia is a form of controlled cellproliferation involving an increase in cell number in a tissue or organ,without significant alteration in structure or function. As but oneexample, endometrial hyperplasia often precedes endometrial cancer.Metaplasia is a form of controlled cell growth in which one type ofadult or fully differentiated cell substitutes for another type of adultcell. Metaplasia can occur in epithelial or connective tissue cells.Atypical metaplasia involves a somewhat disorderly metaplasticepithelium. Dysplasia is frequently a forerunner of cancer, and is foundmainly in the epithelia; it is the most disorderly form ofnon-neoplastic cell growth, involving a loss in individual celluniformity and in the architectural orientation of cells. Dysplasticcells often have abnormally large, deeply stained nuclei, and exhibitpleomorphism. Dysplasia characteristically-occurs where there existschronic irritation or inflammation, and is often found in the cervix,respiratory passages, oral cavity, and gall bladder.

In a preferred embodiment of the invention, a patient in whose DNA isdetected a mutation in the PHIT gene, particularly a deletion, and mostparticularly a homozygous mutation, is thereby determined to have apredisposition to cancer and is treated by administration of a Fhitprotein or functional derivative thereof or nucleic acid encoding thesame (gene therapy).

Alternatively or in addition to the presence of abnormal cell growthcharacterized as hyperplasia, metaplasia, or dysplasia, the presence ofone or more characteristics of a transformed phenotype, or of amalignant phenotype, displayed in vivo or displayed in vitro by a cellsample from a patient, can indicate the desirability ofprophylactic/therapeutic administration of a Therapeutic that promotesFhit function. As mentioned supra, such characteristics of a transformedphenotype include morphology changes, looser substratum attachment, lossof contact inhibition, loss of anchorage dependence, protease release,increased sugar transport, decreased serum requirement, expression offetal antigens, disappearance of the 250,000 dalton cell surfaceprotein, etc. (see also id., at pp. 84-90 for characteristics associatedwith a transformed or malignant phenotype).

In a specific embodiment, leukoplakia, a benign-appearing hyperplasticor dysplastic lesion of the epithelium, or Bowen's disease, a carcinomain situ, are pre-neoplastic lesions indicative of the desirability ofprophylactic intervention.

In another embodiment, fibrocystic disease (cystic hyperplasia, mammarydysplasia, particularly adenosis (benign epithelial hyperplasia)) isindicative of the desirability of prophylactic intervention.

In other embodiments, a patient which exhibits one or more of thefollowing predisposing factors for malignancy is treated byadministration of an effective amount of a Therapeutic: a chromosomaltranslocation associated with a malignancy (e.g., the Philadelphiachromosome for chronic myelogenous leukemia, t(14;18) for follicularlymphoma, etc.), familial polyposis or Gardner's syndrome (possibleforerunners of colon cancer), benign monoclonal gammopathy (a possibleforerunner of multiple myeloma), and a first degree kinship with personshaving a cancer or precancerous disease showing a Mendelian (genetic)inheritance pattern (e.g., familial polyposis of the colon, Gardner'ssyndrome, hereditary exostosis, polyendocrine adenomatosis, medullarythyroid carcinoma with amyloid production and pheochromocytoma,Peutz-Jeghers syndrome, neurofibromatosis of Von Recklinghausen,retinoblastoma, carotid body tumor, cutaneous melanocarcinoma,intraocular melanocarcinoma, xeroderma pigmentosum, ataxiatelangiectasia, Chediak-Higashi syndrome, albinism, Fanconi's aplasticanemia, and Bloom's syndrome; see Robbins and Angell, 1976, BasicPathology, 2d Ed., W.B. Saunders Co., Philadelphia, pp. 112-113) etc.)

In a specific embodiment, a Therapeutic of the invention is administeredto a human patient to prevent progression to breast, colon, lung,stomach or uterine cancer, or melanoma or sarcoma.

5.8.3. Hyperproliferative and Dysproliferative Disorders

In another embodiment of the invention, a Therapeutic that promotes Fhitactivity is used to treat or prevent hyperproliferative or benigndysproliferative disorders. Specific embodiments are directed totreatment or prevention of benign tumors, fibrocystic conditions, andtissue hypertrophy (e.g., prostatic hyperplasia). In specificembodiments, a patient having an intestinal polyp, colon polyp, oresophageal dysplasia is treated by administration of a Therapeutic.

5.8.4. Gene Therapy

In a specific embodiment, nucleic acids comprising a sequence encoding aFhit protein or functional derivative thereof, are administered topromote Fhit function, by way of gene therapy. Gene therapy refers totherapy performed by the administration of a nucleic acid to a subject.

A FHIT polynucleotide may be used in the treatment of various diseasestates associated with chromosome 3p14.2 abnormalities, such as cancers,and/or decreased expression of wild-type FHIT RNA or protein. Byintroducing FHIT gene sequences into cells, gene therapy can be used totreat conditions associated with under-expression of functional FHIT RNAor protein. Accordingly, the present invention provides a method fortreating a disease state associated with a chromosome 3p14.2 abnormalityin mammal suffering from a disease state associated with a chromosome3p14.2 abnormality comprising administering a therapeutically effectiveamount of a nucleic acid encoding a functional Fhit protein to a mammalsuffering from a disease state associated with a chromosome 3p14.2abnormality. In this embodiment of the invention, the nucleic acidproduces its encoded protein that mediates a therapeutic effect bypromoting Fhit function, thereby, e.g., inhibiting tumor or cancerappearance or progression.

Any of the methods for gene therapy available in the art can be usedaccording to the present invention. Exemplary methods are describedbelow.

For general reviews of the methods of gene therapy, see Goldspiel etal., 1993, Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596;Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann.Rev. Biochem. 62:191-217; May, 1993, TIBTECH 11(5):155-215). Methodscommonly known in the art of recombinant DNA technology which can beused are described in Ausubel et al. (eds.), 1993, Current Protocols inMolecular Biology, John Wiley & Sons, NY; and Kriegler, 1990, GeneTransfer and Expression, A Laboratory Manual, Stockton Press, NY.

In a preferred aspect, the Therapeutic comprises a FHIT nucleic acidthat is part of an expression vector that expresses a Fhit protein orfragment or chimeric protein thereof in a suitable host. In particular,such a nucleic acid has a promoter operably linked to the FHIT codingregion, said promoter being inducible or constitutive, and, optionally,tissue-specific. In another particular embodiment, a nucleic acidmolecule is used in which the FHIT coding sequences and any otherdesired sequences are flanked by regions that promote homologousrecombination at a desired site in the genome, thus providing forintrachromosomal expression of the FHIT nucleic acid (Koller andSmithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra etal., 1989, Nature 342:435-438).

Delivery of the nucleic acid into a patient may be either direct, inwhich case the patient is directly exposed to the nucleic acid ornucleic acid-carrying vector, or indirect, in which case, cells arefirst transformed with the nucleic acid in vitro, then transplanted intothe patient. These two approaches are known, respectively, as in vivo orex vivo gene therapy.

In a specific embodiment, the nucleic acid is directly administered invivo, where it is expressed to produce the encoded product. This can beaccomplished by any of numerous methods known in the art, e.g., byconstructing it as part of an appropriate nucleic acid-expression vectorand administering it so that it becomes intracellular, e.g., byinfection using a defective or attenuated retroviral or other viralvector (see U.S. Pat. No. 4,980,286), or by direct injection of nakedDNA, or by use of microparticle bombardment (e.g., a gene gun;Biolistic, Dupont), or coating with lipids or cell-surface receptors ortransfecting agents, encapsulation in liposomes, microparticles, ormicrocapsules, or by administering it in linkage to a peptide which isknown to enter the nucleus, by administering it in linkage to a ligandsubject to receptor-mediated endocytosis (see e.g., Wu and Wu, 1987, J.Biol. Chem. 262:4429-4432) (which can be used to target cell typesspecifically expressing the receptors), etc. In another embodiment, anucleic acid-ligand complex can be formed in which the ligand comprisesa fusogenic viral peptide to disrupt endosomes, allowing the nucleicacid to avoid lysosomal degradation. In yet another embodiment, thenucleic acid can be targeted in vivo for cell specific uptake andexpression, by targeting a specific receptor (see, e.g., PCTPublications WO 92/06180 dated Apr. 16, 1992 (Wu et al.); WO 92/22635dated Dec. 23, 1992 (Wilson et al.); WO92/20316 dated Nov. 26, 1992(Findeis et al.); WO93/14188 dated Jul. 22, 1993 (Clarke et al.), WO93/20221 dated Oct. 14, 1993 (Young)). Alternatively, the nucleic acidcan be introduced intracellularly and incorporated within host cell DNAfor expression, by homologous recombination (Koller and Smithies, 1989,Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra et al., 1989, Nature342:435-438).

In a specific embodiment, a viral vector that contains the FHIT nucleicacid is used. For example, a retroviral vector can be used (see Milleret al., 1993, Meth. Enzymol. 217:581-599). These retroviral vectors havebeen modified to delete retroviral sequences that are not necessary forpackaging of the viral genome and integration into host cell DNA. TheFHIT nucleic acid to be used in gene therapy is cloned into the vector,which facilitates delivery of the gene into a patient. More detail aboutretroviral vectors can be found in Boesen et al., 1994, Biotherapy6:291-302, which describes the use of a retroviral vector to deliver themdr1 gene to hematopoietic stem cells in order to make the stem cellsmore resistant to chemotherapy. Other references illustrating the use ofretroviral vectors in gene therapy are: Clowes et al., 1994, J. Clin.Invest. 93:644-651; Kiem et al., 1994, Blood 83:1467-1473; Salmons andGunzberg, 1993, Human Gene Therapy 4:129-141; and Grossman and Wilson,1993, Curr. Opin. in Genetics and Devel. 3:110-114.

Adenoviruses are other viral vectors that can be used in gene therapy.Adenoviruses are especially attractive vehicles for delivering genes torespiratory epithelia. Adenoviruses naturally infect respiratoryepithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson, 1993,Current Opinion in Genetics and Development 3:499-503 present a reviewof adenovirus-based gene therapy. Bout et al., 1994, Human Gene Therapy5:3-10 demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviruses in gene therapy can be found in Rosenfeld et al., 1991,Science 252:431-434; Rosenfeld et al., 1992, Cell 68:143-155; andMastrangeli et al., 1993, J. Clin. Invest. 91:225-234.

Adeno-associated virus (AAV) has also been proposed for use in genetherapy (Walsh et al., 1993, Proc. Soc. Exp. Biol. Med. 204:289-300.Herpesviruses can also be used.

Another approach to gene therapy involves transferring a gene to cellsin tissue culture by such methods as electroporation, lipofection,calcium phosphate mediated transfection, or viral infection. Usually,the method of transfer includes the transfer of a selectable marker tothe cells. The cells are then placed under selection to isolate thosecells that have taken up and are expressing the transferred gene. Thosecells are then delivered to a patient.

In this embodiment, the nucleic acid is introduced into a cell prior toadministration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including but not limited to transfection, electroporation,microinjection, infection with a viral or bacteriophage vectorcontaining the nucleic acid sequences, cell fusion, chromosome-mediatedgene transfer, microcell-mediated gene transfer, spheroplast fusion,etc. Numerous techniques are known in the art for the introduction offoreign genes into cells (see e.g., Loeffler and Behr, 1993, Meth.Enzymol. 217:599-618; Cohen et al., 1993, Meth. Enzymol. 217:618-644;Cline, 1985, Pharmac. Ther. 29:69-92) and may be used in accordance withthe present invention, provided that the necessary developmental andphysiological functions of the recipient cells are not disrupted. Thetechnique should provide for the stable transfer of the nucleic acid tothe cell, so that the nucleic acid is expressible by the cell andpreferably heritable and expressible by its cell progeny.

The resulting recombinant cells can be delivered to a patient by variousmethods known in the art. In a preferred embodiment, epithelial cellsare injected, e.g., subcutaneously. In another embodiment, recombinantskin cells may be applied as a skin graft onto the patient. Recombinantblood cells (e.g., hematopoietic stem or progenitor cells) arepreferably administered intravenously. The amount of cells envisionedfor use depends on the desired effect, patient state, etc., and can bedetermined by one skilled in the art.

Cells into which a nucleic acid can be introduced for purposes of genetherapy encompass any desired, available cell type, and include but arenot limited to epithelial cells, endothelial cells, keratinocytes,fibroblasts, muscle cells, hepatocytes; blood cells such as Tlymphocytes, B lymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, granulocytes; various stem or progenitorcells, in particular hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, etc.

In a preferred embodiment, the cell used for gene therapy is autologousto the patient.

In an embodiment in which recombinant cells are used in gene therapy, aFHIT nucleic acid is introduced into the cells such that it isexpressible by the cells or their progeny, and the recombinant cells arethen administered in vivo for therapeutic effect. In a specificembodiment, stem or progenitor cells are used. Any stem and/orprogenitor cells which can be isolated and maintained in vitro canpotentially be used in accordance with this embodiment of the presentinvention. Such stem cells include but are not limited to hematopoieticstem cells (HSC), stem cells of epithelial tissues such as the skin andthe lining of the gut, embryonic heart muscle cells, liver stem cells(PCT Publication WO 94/08598, dated Apr. 28, 1994), and neural stemcells (Stemple and Anderson, 1992, Cell 71:973-985).

Epithelial stem cells (ESCs) or keratinocytes can be obtained fromtissues such as the skin and the lining of the gut by known procedures(Rheinwald, 1980, Meth. Cell Bio. 21A:229). In stratified epithelialtissue such as the skin, renewal occurs by mitosis of stem cells withinthe germinal layer, the layer closest to the basal lamina. Stem cellswithin the lining of the gut provide for a rapid renewal rate of thistissue. ESCs or keratinocytes obtained from the skin or lining of thegut of a patient or donor can be grown in tissue culture (Rheinwald,1980, Meth. Cell Bio. 21A:229; Pittelkow and Scott, 1986, Mayo ClinicProc. 61:771). If the ESCs are provided by a donor, a method forsuppression of host versus graft reactivity (e.g., irradiation, drug orantibody administration to promote moderate immunosuppression) can alsobe used.

With respect to hematopoietic stem cells (HSC), any technique whichprovides for the isolation, propagation, and maintenance in vitro of HSCcan be used in this embodiment of the invention. Techniques by whichthis may be accomplished include (a) the isolation and establishment ofHSC cultures from bone marrow cells isolated from the future host, or adonor, or (b) the use of previously established long-term HSC cultures,which may be allogeneic or xenogeneic. Non-autologous HSC are usedpreferably in conjunction with a method of suppressing transplantationimmune reactions of the future host/patient. In a particular embodimentof the present invention, human bone marrow cells can be obtained fromthe posterior iliac crest by needle aspiration (see, e.g., Kodo et al.,1984, J. Clin. Invest. 73:1377-1384). In a preferred embodiment of thepresent invention, the HSCs can be made highly enriched or insubstantially pure form. This enrichment can be accomplished before,during, or after long-term culturing, and can be done by any techniquesknown in the art. Long-term cultures of bone marrow cells can beestablished and maintained by using, for example, modified Dexter cellculture techniques (Dexter et al., 1977, J. Cell Physiol. 91:335) orWitlock-Witte culture techniques (Witlock and Witte, 1982, Proc. Natl.Acad. Sci. USA 79:3608-3612).

In a specific embodiment, the nucleic acid to be introduced for purposesof gene therapy comprises an inducible promoter operably linked to thecoding region, such that expression of the nucleic acid is controllableby controlling the presence or absence of the appropriate inducer oftranscription.

Additional methods that can be adapted for use to deliver a nucleic acidencoding a Fhit protein or functional derivative thereof are describedin Section 5.8.5.

5.8.5. Antagonizing Dominant-Negative FHIT Mutations for Treatment orPrevention of Disorders of Overproliferation

The invention also provides methods of treating or preventing disordersof overproliferation (e.g., cancer, hyperproliferative disorders) inwhich the patient has a hemizygous FHIT mutation (presumably adominant-negative FHIT mutation) by specifically antagonizing(administering an antagonist to) the mutant FHIT gene or protein (andnot wild-type FHIT or Fhit). Hemizygosity for a FHIT mutation can bedetected by observing the presence of both normal and mutant FHIT DNA(e.g., cDNA) or RNA in a sample from a patient, e.g., by methods asdescribed in Sections 5.11 and 6 hereof.

For example, in a specific embodiment, an effective amount of antisenseoligonucleotide that inhibits the expression of the mutant FHIT gene,and not the wild-type FHIT gene, is administered. For example, if thehemizygous FHIT mutation in the patient is a deletion of at least aportion of one or more FHIT exons, the antisense oligonucleotide cancomprise a hybridizable sequence complementary to the junction formed bythe deletion, said junction being present in the mutant FHIT gene butnot the wild-type FHIT gene. Thus, the antisense oligonucleotidecomprises a sequence complementary to contiguous sequences from twoexons not naturally found contiguous in wild-type FHIT cDNA.

In another specific embodiment, an antibody can be used therapeuticallyor prophylactically to specifically antagonize the hemizygous Fhitmutant protein. For example, such an antibody can specifically recognizean epitope in a Fhit deletion mutant formed by the fusion of sequencesnot naturally contiguous in the wild-type Fhit protein. For therapeuticpurposes, a Fhit mutant protein can be used as immunogen to makeanti-Fhit antibodies that neutralize the activity of the Fhit mutantprotein and not wild-type Fhit protein. Accordingly, the presentinvention provides a method for treating a disease state associated witha FHIT abnormality in a mammal suffering from a disease state associatedwith a FHIT abnormality comprising administering a therapeuticallyeffective amount of an anti-Fhit antibody specific to the abnormal FHITgene or protein to a mammal suffering from a disease state associatedwith a FHIT abnormality.

In another specific embodiment, a recombinant nucleic acid consisting ofnon-FHIT sequences flanked by FHIT sequences so as to promote homologousrecombination specifically with a mutant FHIT gene in a patient, isintroduced into the patient, in order to “knock out” (inhibit the effectof) the mutant, particularly where such mutant is believed to be adominant-negative one.

Antisense oligonucleotides are described in further detail below.

5.8.5.1. Antisense Regulation of Mutant FHIT Gene Expression

In a specific embodiment, mutant function of Fhit or FHIT isspecifically inhibited by use of FHIT antisense nucleic acids. Thepresent invention provides the therapeutic or prophylactic use ofnucleic acids of at least six nucleotides that are antisense to a geneor cDNA encoding a mutant Fhit. A FHIT “antisense” nucleic acid as usedherein refers to a nucleic acid capable of hybridizing to a portion of aFHIT RNA (preferably mRNA) or mutant form thereof by virtue of somesequence complementarity (other than to nonspecific sequences such as apolyA tail). The antisense nucleic acid may be complementary to a codingand/or noncoding region of a FHIT mRNA. Such antisense nucleic acidshave utility as Therapeutics that inhibit dominant-negative mutant Fhitfunction, and can be used in the treatment or prevention of disorders asdescribed supra in Section 5.8 and its subsections.

The antisense nucleic acids of the invention can be oligonucleotidesthat are double-stranded or single-stranded, RNA or DNA or amodification or derivative thereof, which can be directly administeredto a cell, or which can be produced intracellularly by transcription ofexogenous, introduced sequences.

The invention further provides pharmaceutical compositions comprising aneffective amount of the FHIT antisense nucleic acids of the invention ina pharmaceutically acceptable carrier, as described in Section 5.1.0.

In another embodiment, the invention is directed to methods forinhibiting the expression specifically of a FHIT mutant nucleic acidsequence in a prokaryotic or eukaryotic cell comprising providing thecell with an effective amount of a composition comprising an FHITantisense nucleic acid of the invention.

The FHIT antisense nucleic acids are of at least six nucleotides and arepreferably oligonucleotides (ranging from 6 to about 50oligonucleotides). In specific aspects, the oligonucleotide is at least10 nucleotides, at least 15 nucleotides, at least 100 nucleotides, or atleast 200 nucleotides. The oligonucleotides can be DNA or RNA orchimeric mixtures or derivatives or modified versions thereof,single-stranded or double-stranded. The oligonucleotide can be modifiedat the base moiety, sugar moiety, or phosphate backbone. Theoligonucleotide may include other appending groups such as peptides, oragents facilitating transport across the cell membrane (see, e.g.,Letsinger et al., 1989, Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556;Lemaitre et al., 1987, Proc. Natl. Acad. Sci. 84:648-652; PCTPublication No. WO 88/09810, published Dec. 15, 1988) or blood-brainbarrier (see, e.g., PCT Publication No. WO 89/10134, published Apr. 25,1988), hybridization-triggered cleavage agents (see, e.g., Krol et al.,1988, BioTechniques 6:958-976) or intercalating agents (see, e.g., Zon,1988, Pharm. Res. 5:539-549).

In a preferred aspect of the invention, a FHIT antisense oligonucleotideis provided, preferably of single-stranded DNA. In a most preferredaspect, such an oligonucleotide comprises a sequence antisense to ajunction of two non-normally contiguous sequences in a FHIT genedeletion mutant, most preferably, of a human FHIT gene mutant. Theoligonucleotide may be modified at any position on its structure withsubstituents generally known in the art.

The FHIT antisense oligonucleotide may comprise at least one modifiedbase moiety which is selected from the group including but not limitedto 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthihe, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

In another embodiment, the oligonucleotide comprises at least onemodified sugar moiety selected from the group including but not limitedto arabinose, 2-fluoroarabinose, xylulose, and hexose.

In yet another embodiment, the oligonucleotide comprises at least onemodified phosphate backbone selected from the group consisting of aphosphorothioate, a phosphorodithioate, a phosphoramidothioate, aphosphoramidate, a phosphordiamidate, a methylphosphonate, an alkylphosphotriester, and a formacetal or analog thereof.

In yet another embodiment, the oligonucleotide is an α-anomericoligonucleotide. An α-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual β-units, the strands run parallel to each other (Gautier et al.,1987, Nucl. Acids Res. 15:6625-6641).

The oligonucleotide may be conjugated to another molecule, e.g., apeptide, hybridization triggered cross-linking agent, transport agent,hybridization-triggered cleavage agent, etc.

Oligonucleotides of the invention may be synthesized by standard methodsknown in the art, e.g. by use of an automated DNA synthesizer (such asare commercially available from Biosearch, Applied Biosystems, etc.).These include techniques for chemically synthesizingoligodeoxyribonucleotides well known in the art such as for examplesolid phase phosphoramidite chemical synthesis. As examples,phosphorothioate oligonucleotides may be synthesized by the method ofStein et al. (1988, Nucl. Acids Res. 16:3209), methylphosphonateoligonucleotides can be prepared by use of controlled pore glass polymersupports (Sarin et al., 1988, Proc. Natl. Acad. Sci. U.S.A.85:7448-7451), etc. Alternatively, RNA molecules may be generated by invitro and in vivo transcription of DNA sequences encoding the antisenseRNA molecule. Such DNA sequences may be incorporated into a wide varietyof vectors which incorporate suitable RNA polymerase promoters such asthe T7 or SP6 polymerase promoters. Alternatively, antisense cDNAconstructs that synthesize antisense RNA constitutively or inducibly,depending on the promoter used, can be introduced stably into celllines.

In a specific embodiment, the PHIT antisense oligonucleotide comprisescatalytic RNA, or a ribozyme (see, e.g., PCT International PublicationWO 90/11364, published Oct. 4, 1990; Sarver et al., 1990, Science247:1222-1225). Ribozymes are enzymatic RNA molecules capable ofcatalyzing the specific cleavage of RNA. The mechanism of ribozymeaction involves sequence specific hybridization of the ribozyme moleculeto complementary target RNA, followed by a endonucleolytic cleavage.Within the scope of the invention are engineered hammerhead motifribozyme molecules that specifically and efficiently catalyzeendonucleolytic cleavage of mutant FHIT RNA sequences.

Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the target molecule for ribozymecleavage-sites which include the following sequences, GUA, GUU and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for predicted structuralfeatures such as secondary structure that may render the oligonucleotidesequence unsuitable. The suitability of candidate targets may also beevaluated by testing their accessibility to hybridization withcomplementary oligonucleotides, using ribonuclease protection assays.

In another embodiment, the oligonucleotide is a2′-O-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res.15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBSLett. 215:327-330).

In an alternative embodiment, the FHIT antisense nucleic acid of theinvention is produced intracellularly by transcription from an exogenoussequence. For example, a vector can be introduced in vivo such that itis taken up by a cell, within which cell the vector or a portion thereofis transcribed, producing an antisense nucleic acid (RNA) of theinvention. Such a vector would contain a sequence encoding the FHITantisense nucleic acid. Such a vector can remain episomal or becomechromosomally integrated, as long as it can be transcribed to producethe desired antisense RNA. Such vectors can be constructed byrecombinant DNA technology methods standard in the art. Vectors can beplasmid, viral, or others known in the art, used for replication andexpression in mammalian cells. Expression of the sequence encoding theFHIT antisense RNA can be by any promoter known in the art to act inmammalian, preferably human, cells. Such promoters can be inducible orconstitutive. Such promoters include but are not limited to: the SV40early promoter region (Bernoist and Chambon, 1981, Nature 290:304-310),the promoter contained in the 3′ long terminal repeat of Rous sarcomavirus (Yamamoto et al., 1980, Cell 22:787-797), the herpes thymidinekinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A.78:1441-1445), the regulatory sequences of the metallothionein gene(Brinster et al., 1982, Nature 296:39-42), etc.

The antisense nucleic acids of the invention comprise a sequencecomplementary to at least a portion of an RNA transcript of a FHIT gene,preferably a human mutant FHIT gene. However, absolute complementarity,although preferred, is not required. A sequence “complementary to atleast a portion of an RNA,” as referred to herein, means a sequencehaving sufficient complementarity to be able to hybridize with the RNA,forming a stable duplex; in the case of double-stranded FRIT antisensenucleic acids, a single strand of the duplex DNA may thus be tested, ortriplex formation may be assayed. The ability to hybridize will dependon both the degree of complementarity and the length of the antisensenucleic acid. Generally, the longer the hybridizing nucleic acid, themore base mismatches with a FHIT RNA it may contain and still form astable duplex (or triplex, as the case may be). One skilled in the artcan ascertain a tolerable degree of mismatch by use of standardprocedures to determine the melting point of the hybridized complex.

The FHIT antisense nucleic acids can be used to treat (or prevent)malignancies or hyperproliferative disorders, of a cell type which hasbeen shown to express mutant FHIT RNA. Malignant, neoplastic, andpre-neoplastic cells which can be tested for such expression include butare not limited to those described supra in Sections 5.8. In a preferredembodiment, a single-stranded DNA antisense FHIT oligonucleotide isused.

Malignant (particularly, tumor) cell types which express FHIT RNA can beidentified by various methods known in the art. Such methods include butare not limited to hybridization with a FHIT-specific nucleic acid(e.g., by Northern hybridization, dot blot hybridization, in situhybridization), observing the ability of RNA from the cell type to betranslated in vitro into Fhit protein, etc. (see the assays describedfor diagnosis in Section 5.11). In a preferred aspect, primary tumortissue from a patient can be assayed for FHIT expression prior totreatment.

Pharmaceutical compositions of the invention, comprising an effectiveamount of a FHIT antisense nucleic acid in a pharmaceutically acceptablecarrier, can be administered to a patient having a malignancy which isof a type that expresses mutant FHIT RNA that is specificallyantagonized by the antisense nucleic acid.

The amount of FHIT antisense nucleic acid which will be effective in thetreatment of a particular disease state or condition will depend on thenature of the disease state or condition, and can be determined bystandard clinical techniques. Where possible, it is desirable todetermine the antisense cytotoxicity of the tumor type to be treated invitro, and then in useful animal model systems prior to testing and usein humans.

In a specific embodiment, pharmaceutical compositions comprising FHITantisense nucleic acids are administered via liposomes, microparticles,or microcapsules. In various embodiments of the invention, it may beuseful to use such compositions to achieve sustained release of the FHITantisense nucleic acids. In a specific embodiment, it may be desirableto utilize liposomes targeted via antibodies to specific identifiabletumor antigens (Leonetti et al., 1990, Proc. Natl. Acad. Sci. USA87:2448-2451; Renneisen et al., 1990, J. Biol. Chem. 265:16337-16342).

In a particular embodiment of the invention, antisense FHIToligonucleotides or anti-Fhit antibodies that specifically antagonize amutant PHIT gene or protein present in a patient, are administered tothe patient in combination with administration to the patient of FHITgene therapy (administration of wild-type Fhit function) or functionalFhit protein or agonists.

5.9. Demonstration of Therapeutic or Prophylactic Utility

The FHIT polynucleotides, FHIT protein products, derivatives and analogsthereof, and antibodies thereto, and antisense nucleic acids of theinvention can be tested in vivo for the desired therapeutic orprophylactic activity. For example, such compounds can be tested insuitable animal model systems prior to testing in humans, including butnot limited to rats, mice, chicken, cows, monkeys, rabbits, etc. For invivo testing, prior to administration to humans, any animal model systemknown in the art may be used.

5.10. Therapeutic/Prophylactic Methods and Compositions

The invention provides methods of treatment and prophylaxis byadministration to a subject of an effective amount of a Therapeutic,i.e., a FHIT nucleic acid, FHIT protein, derivative or analog thereof,or antibody thereto of the present invention. In a preferred aspect, theTherapeutic is substantially purified. The subject is preferably ananimal, including but not limited to animals such as cows, pigs,chickens, etc., and is preferably a mammal, and most preferably human.The subject can be a fetus, child, or adult.

In a specific embodiment, a non-human mammal is the subject.

Formulations and methods of administration that can be employed when theTherapeutic comprises a nucleic acid are described in Sections 5.8.4 and5.8.5.1 above; additional appropriate formulations and routes ofadministration can be selected from among those described hereinbelow.

Various delivery systems are known and can be used to administer aTherapeutic of the invention, e.g., encapsulation in liposomes,microparticles, microcapsules, expression by recombinant cells,receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol.Chem. 262:4429-4432), construction of a therapeutic nucleic acid as partof a retroviral or other vector, etc. Methods of introduction includebut are not limited to intradermal, intramuscular, intraperitoneal,intravenous, subcutaneous, intranasal, and oral routes. The compoundsmay be administered by any convenient route, for example by infusion orbolus injection, by absorption through epithelial or mucocutaneouslinings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and maybe administered together with other biologically active agents.Administration can be systemic or local. In addition, it may bedesirable to introduce the pharmaceutical compositions of the inventioninto the central nervous system by any suitable route, includingintraventricular and intrathecal injection; intraventricular injectionmay be facilitated by an intraventricular catheter, for example,attached to a reservoir, such as an Ommaya reservoir.

In a specific embodiment, it may be desirable to administer thepharmaceutical compositions of the invention locally to the area in needof treatment; this may be achieved by, for example, and not by way oflimitation, local infusion during surgery, topical application, e.g., inconjunction with a wound dressing after surgery, by injection, by meansof a catheter, by means of a suppository, or by means of an implant,said implant being of a porous, non-porous, or gelatinous material,including membranes, such as sialastic membranes, or fibers. In oneembodiment, administration can be by direct injection at the site (orformer site) of a malignant tumor or neoplastic or pre-neoplastictissue.

In a specific embodiment where the Therapeutic is a nucleic acidencoding a protein therapeutic, the nucleic acid can be administered invivo to promote expression of its encoded protein, by constructing it aspart of an appropriate nucleic acid expression vector and administeringit so that it becomes intracellular, e.g., by use of a retroviral vector(see U.S. Pat. No. 4,980,286), or by direct injection, or by use ofmicroparticle bombardment (e.g., a gene gun; Biolistic, Dupont), orcoating with lipids or cell-surface receptors or transfecting agents, orby administering it in linkage to a homeobox-like peptide which is knownto enter the nucleus (see e.g., Joliot et al., 1991, Proc. Natl. Acad.Sci. USA 88:1864-1868), etc. Alternatively, a nucleic acid therapeuticcan be introduced intracellularly and incorporated within host cell DNAfor expression, by homologous recombination.

The present invention also provides pharmaceutical compositions. Suchcompositions comprise a therapeutically effective amount of atherapeutic, and a pharmaceutically acceptable carrier or excipient.Such a carrier includes but is not limited to saline, buffered saline,dextrose, water, glycerol, ethanol, and combinations thereof. Thecarrier and composition can be sterile. The formulation should suit themode of administration.

The composition, if desired, can also contain minor amounts of wettingor emulsifying agents, or pH buffering agents. The composition can be aliquid solution, suspension, emulsion, tablet, pill, capsule, sustainedrelease formulation, or powder. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc.

In a preferred embodiment, the composition is formulated in accordancewith routine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lignocaine to ease pain at the siteof the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The Therapeutics of the invention can be formulated as neutral or saltforms. Pharmaceutically acceptable salts include those formed with freeamino groups such as those derived from hydrochloric, phosphoric,acetic, oxalic, tartaric acids, etc., and those formed with freecarboxyl groups such as those derived from sodium, potassium, ammonium,calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

The amount of the Therapeutic of the invention which will be effectivein the treatment of a particular disorder or condition will depend onthe nature of the disorder or condition, and can be determined bystandard clinical techniques. In addition, in vitro assays mayoptionally be employed to help identify optimal dosage ranges. Theprecise dose to be employed in the formulation will also depend on theroute of administration, and the seriousness of the disease or disorder,and should be decided according to the judgment of the practitioner andeach patient's circumstances. However, suitable dosage ranges forintravenous administration are generally about 20-500 micrograms ofactive compound per kilogram body weight. Suitable dosage ranges forintranasal administration are generally about 0.01 pg/kg body weight to1 mg/kg body weight. Effective doses may be extrapolated fromdose-response curves derived from in vitro or animal model test systems.

Suppositories generally contain active ingredient in the range of 0.5%to 10% by weight; oral formulations preferably contain 10% to 95% activeingredient.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Optionally associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration.

5.11. Diagnostic Uses

A FHIT polynucleotide and nucleic acids complementary thereto, its Fhitprotein product, fragments thereof, and antibodies thereto can be usedfor diagnostic purposes for disorders involving overproliferation ofcells, as well as other disorders associated with chromosomaltranslocations and inversions or molecular abnormalities associated withthe FHIT gene, and/or decreased expression of wild-type FHIT RNA orprotein.

Such molecules can also be used in diagnostic assays, such asimmunoassays, to detect, prognose, diagnose, or monitor variousconditions, diseases, and disorders associated with expression of mutantFHIT transcripts or monitor the treatment thereof. Accordingly, inspecific embodiments, cancer or premalignant changes orhyperproliferative or benign dysproliferative disorders in tissue isdiagnosed by detecting the presence of one or more mutant FHITtranscripts, alone or in combination with a decrease in expression ofwild-type FHIT transcript, in patient samples relative to FHITexpression in an analogous non-diseased sample (from the patient oranother person, as determined experimentally or as is known as astandard level in such samples). For diagnostic purposes, a FHITpolynucleotide may be used to detect mutant FHIT gene expression indisease states.

The subject, or patient, is an animal, e.g., a mammal, and is preferablyhuman, and can be a fetus, child, or adult.

As illustrated infra, the FHIT gene sequence is associated with cancers,particularly associated with translocations and deletions within theFHIT gene. In specific embodiments, diseases and disorders involvingoverproliferation of cells can be diagnosed, or their suspected presencecan be screened for, or a predisposition to develop such disorders canbe detected, by detecting decreased levels of wild-type Fhit protein,wild-type FHIT RNA, or Fhit functional activity, or by detectingmutations in FHIT RNA, DNA, cDNA, or protein (e.g., translocations ordeletions in FHIT nucleic acids, truncations in the FHIT gene orprotein, changes in nucleotide or amino acid sequence relative towild-type Fhit) that cause decreased expression or activity of Fhit or adominant-negative effect. Such diseases and disorders include but arenot limited to those described in Section 5.8 and its subsections. Byway of example, levels of Fhit protein can be detected by immunoassay,levels of FHIT RNA can be detected by hybridization assays (e.g.,Northern blots, dot blots) or RT-PCR, translocations, deletions, andpoint mutations in FHIT nucleic acids can be detected by Southernblotting, RFLP analysis, PCR of cDNA using primers that preferablygenerate a fragment spanning at least most of the FHIT gene, sequencingof the FHIT genomic DNA or cDNA obtained from the patient, etc.

In a preferred embodiment, levels of FHIT mRNA (or cDNA) or protein in apatient sample are detected or measured or analyzed by size and/orsequence, in which aberrant levels, size or sequence indicate that thesubject has, or has a predisposition to developing, a malignancy orhyperproliferative disorder; in which the decreased levels are relativeto the levels present in an analogous sample from a portion of the bodyor from a subject not having the malignancy or hyperproliferativedisorder, as the case may be.

FHIT gene sequences may be used diagnostically for the detection ofdiseases states resulting from chromosomal or molecular abnormalities,e.g., translocations, inversions and deletions, involving the FHIT gene.Nucleic acids comprising FHIT nucleotide sequences of at least 8nucleotides, at least 15 nucleotides, at least 25 nucleotides, at least50 nucleotides, at least 100 nucleotides, at least 200 nucleotides, atleast 300 nucleotides, and preferably less than 500 nucleotides, and thenucleic acids described in Section 5.1, may be used as probes inhybridization assays for the detection and measurement of FHIT genesequences. Nucleic acids of not more than 5 kilobases, of not more than10 kilobases, not more than 25 kilobases, not more than 50 kilobases ornot more than 70 kilobases which are hybridizable to a FHIT gene, cDNA,or complementary strand can be used as probes in hybridization assaysfor the detection and measurement of FHIT nucleotide sequences. As anexample, the FHIT DNA sequence may be used in hybridization assays,e.g., Southern or Northern analysis, including in situ hybridizationassays, of patient's samples to diagnose abnormalities of FHITexpression. Hybridization assays can be used to detect, prognose,diagnose, or monitor malignancies, associated with aberrant changes inFHIT expression and/or activity as described supra. In particular, sucha hybridization assay is carried out by a method comprising contacting asample containing nucleic acid with a nucleic acid probe capable ofhybridizing to FHIT DNA (e.g., cDNA) or RNA, under conditions such thathybridization can occur, and detecting or measuring any resultinghybridization. In particular, hybridization assays can be used to detectthe presence of abnormalities associated with expression of mutant FHITmRNA, by hybridizing mRNA or cDNA from a patient sample to a FHIT probe,and analyzing by size and/or sequence the resulting hybridized nucleicacids. For example, assays which can be used include, but are notlimited to Northern blots, dot blots, etc. A particular hybridizationassay is Northern blot analysis of a patient sample using FHIT geneprobes of at least 15 nucleotides up to the full length cDNA sequenceshown in FIG. 2A. Another hybridization assay is in situ hybridizationanalysis of a patient sample using anti-FHIT antibodies or FHITnucleotide hybridization probes. Such techniques are well known in theart, and are in fact the basis of many commercially available diagnostickits.

In a specific embodiment, cancer or other disorder of celloverproliferation (e.g., those described in Sections 5.8.1-5.8.3 above),is diagnosed or prognosed by detecting a mutation in the FHIT gene orits produced RNA in a sample derived from a patient. The mutation can bea translocation, deletion, insertion or substitution/point mutation. Ina preferred embodiment, the mutation is a deletion of all or a portionof at least one coding exon (i.e., exon 5, 6, 7, 8 or 9), preferablyexon 5 or exon 8. In a preferred embodiment, the deletion is ahomozygous deletion. In another embodiment, the mutation is a mutationthat causes a frameshift upstream of exon 8, or otherwise causes a lackof the wild-type open reading frame (ORF) of exon 8 in the patient'sFHIT RNA.

In other specific embodiments, the mutation is a deletion of FHIT exons4-6 resulting in a fusion of exon 3 sequences to exon 7 sequences in aFHIT RNA or cDNA, or the mutation is a deletion of FHIT exons 4-8resulting in a fusion of exon 3 sequences to exon 9 sequences in a FHITRNA or cDNA.

In another particular embodiment, the mutation that is detected is aninsertion into a coding region of the FHIT gene or an insertiondownstream of exon 4, or an insertion in the 5′ noncoding region betweenexon 4 and 5. In a specific embodiment, the mutation in the FHIT genecoding sequence is detected by detecting an aberrant sized FHIT cDNA ormRNA from the subject (i.e., FHIT RNA or cDNA that has a different sizethan the wild-type FHIT RNA (that is present or expected to be presentin normal individuals not having or pre-disposed to a cancer associatedwith a FHIT mutation, e.g., the ˜1.1 kb transcript)).

In another embodiment, diagnosis or prognosis is carried out bydetecting an aberrant sized FHIT cDNA or mRNA from the subject as wellas the loss of one FHIT allele in the subject.

Polynucleotide sequences of FHIT consisting of at least 8 to 25nucleotides that are useful as primers in primer dependent nucleic acidamplification methods may be used for the detection of mutant FHITgenomic or RNA sequences in patient samples. Primer dependent nucleicacid amplification methods useful in the present invention include, butare not limited to, polymerase chain reaction (PCR), competitive PCR,cyclic probe reaction, and ligase chain reaction. Such techniques arewell known by those of skill in the art. A preferred nucleic acidamplification method of the present invention is reverse transcriptasePCR (RT-PCR) (Siebert et al., 1992, Nature 359:557-558).

In a particular embodiment of the present invention, each primer of apair of primers for use in a primer dependent nucleic acid amplificationmethod is selected from a different exon of the genomic FHIT nucleotidesequences. For example, if one primer of a pair or primers is selectedfrom exon 1 of the FHIT genomic sequence, the second primer will beselected from exon 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the FHIT genomicsequence. As another example, if one primer of a pair of primers isselected from exon 2 of the FHIT genomic sequence, the second primerwill be selected from exon 1, 3, 4, 5, 6, 7, 8, 9 or 10 of the FHITgenomic sequence. Resulting amplified genomic nucleotide sequences willcontain amplified intron sequences and will be of a larger size thanamplified cDNA nucleotide sequences that will not contain amplifiedintron sequences. Similarly, amplified cDNA sequences having a deletionmutation can be distinguished from amplified wild-type sequences due tothe size difference of the resulting amplified sequences (the deletionmutant will generate a shorter amplified fragment). For amplification ofcDNA nucleotide sequences, the primer sequences should be selected fromexons sequences that are sufficiently far enough apart to provide adetectable amplified nucleotide sequence.

In a specific embodiment, cancer or other disorder of cell proliferationor a predisposition thereto is detected or diagnosed in a subject bydetecting mutation(s) within the FHIT gene as follows: A samplecontaining RNA of tissue or cells of a patient is obtained, and the RNAis reverse-transcribed into cDNA by methods commonly known in the art;preferably this step is followed by amplifying fragments comprising FHITcoding sequences within the cDNA, and detecting one or more mutation(s)within the FHIT coding sequences within the amplified fragment. Theamplification can be by any suitable methods known in the art, and ispreferably done by polymerase chain reaction (PCR). RT-PCR is preferreddue to the great size (>500 kb) of the FHIT gene in the genome, whichrenders one unable to amplify a single fragment containing most of theFHIT exons from a genomic sample, whereas amplification of such afragment is readily accomplished from a cDNA sample. The primers for usein PCR are upstream and downstream primers that prime synthesis by apolymerase toward each other, and are preferably in the range of 8-35nucleotides, preferably separated by in the range of 10-2,000nucleotides in the FHIT mRNA. In a preferred embodiment, each primercomprises a hybridizable sequence contained within an exon of the FHITgene or within 200 nucleotides flanking (5′ or 3′ to) an exon of theFHIT gene. In a specific embodiment, the first primer hybridizes 5′ toexon 5 (preferably containing sequences of exon 4 or 5′ thereto) and thesecond primer hybridizes on the other strand 5′ to the intron betweenexons 5 and 6 (such that an amplified fragment from wild-type FHIT cDNAwould contain exon 5). In another specific embodiment, the second primerhybridizes on the other strand 5′ to exon 6. In other specificembodiments, the first and second primers respectively hybridize onopposite strands 5′ to the 3′ terminus of exon 4 and 5′ to exon 8; 5′ tothe 3′ terminus of exon 4 and 5′ to exon 9; and 5′ to exon 1 and 5′ toexon 10, such that the resulting amplified fragment would contain theexon sequences normally present between where the primers hybridizeshould they be present in the cDNA. Thus, for example, in the foregoingexamples, the PCR primer pairs are adapted to amplify a fragment ofwild-type FHIT cDNA comprising FHIT exon 5, exon 5 plus exon 6,sequences between the 3′ terminus of exon 4 and exon 8, sequencesbetween the 3′ terminus of exon 4 and exon 9, and exons 1 through 10,respectively. The presence of one or more mutations in the cDNA can bedetected by detecting an aberrantly sized (preferably amplified)fragment (compared to those fragment(s) produced by a wild-type FHITtranscript), e.g., by subjecting the cDNA to size separation such as byagarose gel electrophoresis or column chromatography. In a preferredembodiment, the presence of one or more mutations in the cDNA isdetected by sequencing of the cDNA, or more preferably, of the isolatedfragments amplified from the cDNA. The amplified fragments can beisolated by methods known in the art, e.g., agarose gel electrophoresisand recovery from the gel band and/or column chromatography. Suchsequencing can be carried out by standard methods commonly known in theart, and can be automated or manual.

In yet another specific embodiment, mutation(s) in the FHIT gene or mRNAfrom a patient can be detected by other methods commonly known in theart, e.g., Northern hybridization. By way of example but not limitation,RNA from a patient's tissue is separated by gel electrophoresis,transferred to a filter or other solid phase, and hybridized to labelledDNA probes. The hybridized RNAs are then visualized by detecting thelabel. Preferably, numerous DNA probes are used, from different portionsof the FHIT cDNA.

In another embodiment, Southern hybridization can be used to detectgross mutations in FHIT DNA. For example, genomic DNA isolated from apatient, separated by gel electrophoresis, transferred to a filter orother solid phase, and hybridized with a FHIT probe (e.g., anoligonucleotide containing a FHIT gene sequence, affixed to a detectablelabel). Preferably, a multiplicity of FHIT probes are used, hybridizableto sequences within each of the coding exons, and particularlypreferably, including probe(s) hybridizable to sequences within exon 5.

In another embodiment, a translocation within the FHIT gene is detectedby methods commonly known in the art. For example, in a preferredembodiment, a sample comprising PHIT genomic DNA, or, preferably FHITcDNA (e.g., cDNA of total polyA mRNA) from a patient is subjected to PCRby use of primers that prime synthesis across the suspectedtranslocation junction. For example, one primer can have a sequencehybridizable to chromosome 3 (preferably within the FHIT gene upstreamof exon 4, e.g., a sequence within exon 1, 2 or 3) and one primer canhave a sequence hybridizable to chromosome 8 (downstream of thetranslocation event); amplification of a fragment indicates the presenceof a translocation between chromosomes 3 and 8. Additionally oralternatively performing PCR by priming with primers, each having asequence within the FHIT gene (see e.g., description supra regardingprimers for RT-PCR) will result in an amplified fragment only if atleast one FHIT allele contains the primer sequences undisrupted by atranslocation event in between them.

Detection of homozygous mutations (mutations in both alleles) in FHITgenes are deemed more severe indicators of the presence of, or apredisposition to, cancer than hemizygous mutations (of one allele) inFHIT genes.

As used herein, patient samples which can be used include, but are notlimited to, fresh or frozen tissue samples, which can be used in in situhybridization assays; cell or tissue from biopsies and, in general,patient samples containing nucleic acid, which can be used in assaysthat measure or quantitate or analyze FHIT nucleic acid.

The FHIT gene sequences of the present invention may be useddiagnostically for the detection of chromosome 3p14.2 abnormalities, inparticular, translocations with chromosome 8, and deletions.Accordingly, the present invention provides a process for detecting atarget sequence indicative of or including a chromosome 3p14.2abnormality in a sample, comprising the steps of amplifying the targetsequence in the sample using a first primer of 8 to 25 nucleotides,preferably 18-25 nucleotides, complementary to the nucleotide sequenceof SEQ ID NO: 1, and a second primer complementary to a region telomericor centromeric to the FHIT gene and detecting any resulting amplifiedtarget sequence in which the presence of the amplified target sequenceis indicative of the abnormality. The present invention also provides amethod of diagnosing a malignancy associated with chromosome 3p14.2abnormalities in a patient comprising, detecting said chromosome 3p14.2abnormality according to the method above in which the presence of anamplified target sequence indicative of a mutant FHIT transcriptindicates the presence of a cancer or precancerous condition in thepatient. The resultant amplified target sequence can be detected on gelelectrophoresis and compared with a normal sample or standard that doesnot contain a chromosome 3p14.2 abnormality. The amplification ofgenomic DNA target sequences may require generating long PCR products.PCR techniques for generating long PCR products are described in Science(1994) 263:1564-1565; PCR kits for generating long PCR products areavailable from Perkin Elmer and Takara Shuzo Co., Ltd. The presentinvention also provides a method for detecting a target nucleotidesequence indicative of or including at least a portion of a chromosome3p14.2 abnormality (thereby indicative of the presence of or apredisposition to a disorder of cell overproliferation) in a nucleicacid sample, comprising the steps of hybridizing the sample with anucleic acid probe of not more than 10 kilobases, comprising FHIT cDNAsequences selected from among at least exon 1, 2, 3 or 4 and selectedfrom among at least exon 7, 8 or 9, or a sequence absolutelycomplementary thereto, and detecting or measuring the amount of anyresulting hybridization between the probe and the target sequence withinthe sample. Alternatively, the probe comprises at least 310 contiguousnucleotides of a FHIT cDNA, or at least 266 contiguous nucleotides ofFHIT cDNA coding sequences. The resultant hybridization between theprobe and the target sequence within the sample can be detected usinggel electrophoresis and can be compared to a target sequence from anormal sample or standard that does not contain the abnormality. Thepresent invention also provides a method of diagnosing a malignancyassociated with a FHIT abnormality in a patient comprising detectingsaid FHIT abnormality according to the method above in which thepresence of the amplified target sequence indicates the presence of amalignancy in the patient. Absolute complementarity between ahybridization probe and a target sequence, although preferred, is notrequired. A sequence “complementary to at least a portion of”, asreferred to herein, means a sequence having sufficient complementarityto be able to hybridize with the nucleic acid, forming a stablehybridization complex. The ability to hybridize will depend on both thedegree of complementarity and the length of the nucleic acid. Generally,the longer the hybridizing nucleic acid, the more base mismatches with aFHIT RNA it may contain and still form a stable duplex (or triplex, asthe case may be). One skilled in the art can ascertain a tolerabledegree of mismatch by use of standard procedures to determine themelting point of the hybridized complex.

An additional aspect of the present invention relates to diagnostic kitsfor the detection or measurement of FHIT gene sequences and FHITprotein. Kits for diagnostic use are provided, that comprise in one ormore containers an anti-Fhit antibody, and, optionally, a labeledbinding partner to the antibody. Alternatively, the anti-Fhit antibodycan be labeled (with a detectable marker, e.g., a chemiluminescent,enzymatic, fluorescent, or radioactive moiety). A kit is also providedthat comprises in one or more containers a nucleic acid probe capable ofhybridizing to FHIT RNA. Accordingly, the present invention provides adiagnostic kit comprising, in a container a compound comprising a probeof not more than 10 kilobases and comprising FHIT cDNA sequencescomprising at least one of exon 1, 2, 3 or 4 and at least one of exon 7,8 or 9; or its complement. Alternatively, the probe comprises at least310 contiguous nucleotides of a FHIT cDNA, or at least 266 contiguousnucleotides of FHIT cDNA coding sequences. Alternatively, the presentinvention provides a diagnostic kit comprising, in one or morecontainers, a pair of primers of at least 8-35, preferably 8-25,nucleotides in which at least one of said primers is hybridizable to SEQID NO: 1 or its complement and wherein said primers are capable ofpriming cDNA synthesis in an amplification reaction. In a specificembodiment, a kit can comprise in one or more containers a pair ofprimers (e.g., each in the size range of 8-35 nucleotides) that arecapable of priming amplification [e.g., by polymerase chain reaction(see e.g., Innis et al., 1990, PCR Protocols, Academic Press, Inc., SanDiego, Calif.), ligase chain reaction (see EP 320,308) use of Qβreplicase, cyclic probe reaction, or other methods known in the art]under appropriate reaction conditions of at least a portion of a FHITnucleic acid. The present invention also provides a diagnostic kit inwhich at least one of the primers is hybridizable to SEQ ID NO: 1 or itscomplement and in which one of the primers is hybridizable to a DNAsequence located telomeric or centromeric to the FHIT gene. In anotherembodiment, the kit comprises a primer pair such as described supra foruse in diagnostic assays. In a specific embodiment, one of the foregoingcompounds of the container can be detectably labeled. A kit canoptionally further comprise in a container a predetermined amount of apurified Fhit protein or nucleic acid, e.g., for use as a standard orcontrol.

The amplification reaction of the present invention may be a polymerasechain reaction, competitive PCR and competitive reverse-transcriptasePCR (Clementi et al., 1994, Genet Anal Tech Appl 11(1):1-6 and Siebertet al., 1992, Nature 359:557-558); cyclic probe reaction, which allowsfor amplification of a target sequence using a hybrid RNA/DNA probe andRNase (ID Biomedical); ligase chain reaction (Wu et al., 1989, Genomics4:560-569). In a particular embodiment, the chromosomal abnormalityassociated with a FHIT locus can be detected as described in PCTPublication No. WO92/19775, dated Nov. 12, 1992. In a specificembodiment, the FHIT probe used in a hybridization assay is detectablylabeled. Such a label can be any known in the art including, but notlimited to, radioactive labels, fluorescent labels, biotin,chemiluminescent labels, etc.

In a specific embodiment in which the assay used employs primers, atleast one primer can be detectably labeled. In another embodiment, oneof a primer pair is attached to a moiety providing for capture, e.g., amagnetic bead.

Anti-FHIT antibodies may be generated and used diagnostically to detectthe presence of mutant Fhit protein in patient samples, and/or theabsence of wild-type Fhit protein, thereby identifying disease statesassociated with chromosome 3p14.2 abnormalities such as disorders ofcell overproliferation.

For example, in one embodiment, where one is detecting or measuringmutant Fhit protein by assaying for binding to anti-Fhit antibody,various immunoassays known in the art can be used, including but notlimited to competitive and non-competitive assay systems usingtechniques such as radioimmunoassays, ELISA (enzyme linked immunosorbentassay), “sandwich” immunoassays, immunoradiometric assays, gel diffusionprecipitin reactions, immunodiffusion assays, in situ immunoassays(using colloidal gold, enzyme or radioisotope labels, for example),western blots, in situ hybridizations, precipitation reactions,agglutination assays (e.g., gel agglutination assays, hemagglutinationassays), complement fixation assays, immunofluorescence assays, proteinA assays, and immunoelectrophoresis assays, etc. In one embodiment,antibody binding is detected by detecting a label on the primaryantibody. In another embodiment, the primary antibody is detected bydetecting binding of a secondary antibody or reagent to the primaryantibody. In a further embodiment, the secondary antibody is labelled.Many means are known in the art for detecting binding in an immunoassayand are within the scope of the present invention. In particular, suchan immunoassay is carried out by a method comprising contacting a samplederived from a patient with an anti-Fhit antibody under conditions suchthat immunospecific binding can occur, and detecting or measuring theamount of any immunospecific binding by the antibody. In a specificembodiment, antibody to a Fhit protein can be used to assay a patienttissue or serum sample for the presence of a FHIT protein where anincreased level of FHIT protein is an indication of a diseasedcondition. In one embodiment of the present invention, the FHIT proteinis detected or measured by immunocytochemistry of a patient sample. Inanother embodiment, assays to measure the levels of FHIT protein or RNAcan be used to monitor therapy of disease associated with increasedexpression of FHIT. For example, a decrease in levels of FHIT RNA orprotein after therapy, relative to the level found before therapy, maybe indicative of a favorable response to therapy. An increase in suchlevels after therapy may be indicative of a poor response to therapy.

For detection of Fhit protein sequences, a diagnostic kit of the presentinvention comprises, in one or more containers, an anti-Fhit antibodywhich optionally can be detectably labeled. In a different embodiment,the kit can comprise in a container, a labeled specific binding portionof an antibody. As used herein, the term detectable label refers to anylabel which provides directly or indirectly a detectable signal andincludes, for example, enzymes, radiolabelled molecules, fluorescentmolecules, particles, chemiluminesors, enzyme substrates or cofactors,enzyme inhibitors, or magnetic particles. Examples of enzymes useful asdetectable labels in the present invention include alkaline phosphataseand horse radish peroxidase. A variety of methods are available forlinking the detectable labels to proteins of interest and include forexample the use of a bifunctional agent, such as,4,4′-difluoro-3,3′-dinitro-phenylsulfone, for attaching an enzyme, forexample, horse radish peroxidase, to a protein of interest. The attachedenzyme is then allowed to react with a substrate yielding a reactionproduct which is detectable. The present invention provides a method fordetecting a Fhit protein in a patient sample, comprising, contacting thepatient sample with an anti-Fhit antibody under conditions such thatimmunospecific binding can occur, and detecting or measuring the amountof any immunospecific binding by the antibody. The method preferablyalso comprises subjecting the protein to size fractionation and/orsequence determination.

Samples can be any sample from a patient containing FHIT protein, e.g.,tissue sections.

In diagnosing disease states, the functional activity of Fhit proteins,derivatives and analogs may be assayed by various methods. Accordingly,the present invention also provides a method of diagnosing a malignancyor other disorder associated with chromosome 3p14.2 (FHIT) abnormalitiesin a patient comprising, detecting expression of a mutant Fhit proteinin a sample from the patient, in which the presence of a mutant Fhitprotein indicates the presence of a malignancy or other disorderassociated with FHIT abnormalities in the patient.

In a specific embodiment of the invention, prenatal diagnosis of adisorder of cell overproliferation or a predisposition thereto, iscarried out. For example, one can first obtain tissue (e.g., bloodcells) from an expectant parent. If one or more of the expectant parentshave a FHIT mutation, thus indicating possible inheritance of thismutation by the offspring, amniocentesis or some other method of fetaltissue sampling can then be carried out to obtain fetal cells which canthen be tested for the presence of FHIT mutant DNA or RNA or protein bymethods as described above (e.g., RT-PCR to detect mutant FHIT RNA).

In another embodiment, the levels of FHIT protein or RNA expression maybe used to stage or monitor disease, with the appearance of or anincrease in mutant Fhit protein or RNA expression, and/or a decrease ofor loss in wild-type Fhit protein or RNA expression, relative to thatpresent in a sample derived from the subject at an earlier time,indicates disease progression.

The ability of antibodies, peptides or other molecules to modulate theeffect of Fhit protein on disease states may be monitored. For example,the expression of FHIT gene sequences or Fhit protein sequences may bedetected as described, supra, both before and after administration of atherapeutic composition, e.g., comprising a FHIT nucleotide sequence,Fhit protein sequence, derivative or analog thereof, or antibodythereto, or antisense nucleic acid of the present invention.

In another embodiment, presence of FHIT mutation(s), particularlyhomozygous ones, can be used as indicators of adverse outcome to therapyor recurrence of the disorder in patients with disorders of celloverproliferation.

Other methods will be known to the skilled artisan and are within thescope of the invention.

5.12. Screening for Fhit Agonists and Antagonists

FHIT nucleic acids, proteins, and derivatives also have uses inscreening assays to detect molecules that specifically bind to FHITnucleic acids, proteins, or derivatives and thus have potential use asagonists or antagonists of Fhit, in particular, molecules that thusaffect cell proliferation. In a preferred embodiment, such assays areperformed to screen for molecules with potential utility as anti-cancerdrugs or lead compounds for drug development. The invention thusprovides assays to detect molecules that specifically bind to FHITnucleic acids, proteins, or derivatives. For example, recombinant cellsexpressing FHIT nucleic acids can be used to recombinantly produce Fhitproteins in these assays, to screen for molecules that bind to a Fhitprotein. Molecules (e.g., putative binding partners of Fhit) arecontacted with the Fhit protein (or fragment thereof) under conditionsconducive to binding, and then molecules that specifically bind to theFhit protein are identified. Similar methods can be used to screen formolecules that bind to Fhit derivatives or nucleic acids. Methods thatcan be used to carry out the foregoing are commonly known in the art.

In a specific embodiment of the present invention, a Fhit protein and/orcell line that expresses a Fhit protein can be used to screen forantibodies, peptides, or other molecules that bind to the FHIT proteinand thus may act as agonists or antagonists of FHIT protein. Forexample, anti-Fhit antibodies capable of neutralizing the activity of adominant-negative mutant Fhit protein may be used to inhibit or preventa disease state associated with cell overproliferation such as cancer.

Screening of organic or peptide libraries with recombinantly expressedmutant Fhit protein may be useful for identification of therapeuticmolecules that function to inhibit the activity of mutant Fhit protein.Screening against wild-type Fhit protein can then be carried out toselect for antagonists specific to the mutant Fhit protein, i.e., thatdo not inhibit (or bind) the wild-type Fhit protein. Synthetic andnaturally occurring products can be screened in a number of ways deemedroutine to those of skill in the art.

By way of example, diversity libraries, such as random or combinatorialpeptide or nonpeptide libraries can be screened for molecules thatspecifically bind to Fhit. Many libraries are known in the art that canbe used, e.g., chemically synthesized libraries, recombinant (e.g.,phage display libraries), and in vitro translation-based libraries.

Examples of chemically synthesized libraries are described in Fodor etal., 1991, Science 251:767-773; Houghten et al., 1991, Nature 354:84-86;Lam et al., 1991, Nature 354:82-84; Medynski, 1994, Bio/Technology12:709-710; Gallop et al., 1994, J. Medicinal Chemistry 37(9):1233-1251;Ohlmeyer et al., 1993, Proc. Natl. Acad. Sci. USA 90:10922-10926; Erb etal., 1994, Proc. Natl. Acad. Sci. USA 91:11422-11426; Houghten et al.,1992, Biotechniques 13:412; Jayawickreme et al., 1994, Proc. Natl. Acad.Sci. USA 91:1614-1618; Salmon et al., 1993, Proc. Natl. Acad. Sci. USA90:11708-11712; PCT Publication No. WO 93/20242; and Brenner and Lerner,1992, Proc. Natl. Acad. Sci. USA 89:5381-5383.

Examples of phage display libraries are described in Scott and Smith,1990, Science 249:386-390; Devlin et al., 1990, Science, 249:404-406;Christian, R. B., et al., 1992, J. Mol. Biol. 227:711-718); Lenstra,1992, J. Immunol. Meth. 152:149-157; Kay et al., 1993, Gene 128:59-65;and PCT Publication No. WO 94/18318 dated Aug. 18, 1994.

In vitro translation-based libraries include but are not limited tothose described in PCT Publication No. WO 91/05058 dated Apr. 18, 1991;and Mattheakis et al., 1994, Proc. Natl. Acad. Sci. USA 91:9022-9026.

By way of examples of nonpeptide libraries, a benzodiazepine library(see e.g., Bunin et al., 1994, Proc. Natl. Acad. Sci. USA 91:4708-4712)can be adapted for use. Peptoid libraries (Simon et al., 1992, Proc.Natl. Acad. Sci. USA 89:9367-9371) can also be used. Another example ofa library that can be used, in which the amide functionalities inpeptides have been permethylated to generate a chemically transformedcombinatorial library, is described by Ostresh et al. (1994, Proc. Natl.Acad. Sci. USA 91:11138-11142).

Screening the libraries can be accomplished by any of a variety ofcommonly known methods. See, e.g., the following references, whichdisclose screening of peptide libraries: Parmley and Smith, 1989, Adv.Exp. Med. Biol. 251:215-218; Scott and Smith, 1990, Science 249:386-390;Fowlkes et al., 1992; BioTechniques 13:422-427; Oldenburg et al., 1992,Proc. Natl. Acad. Sci. USA 89:5393-5397; Yu et al., 1994, Cell76:933-945; Staudt et al., 1988, Science 241:577-580; Bock et al., 1992,Nature 355:564-566; Tuerk et al., 1992, Proc. Natl. Acad. Sci. USA89:6988-6992; Ellington et al., 1992, Nature 355:850-852; U.S. Pat. No.5,096,815, U.S. Pat. No. 5,223,409, and U.S. Pat. No. 5,198,346, all toLadner et al.; Rebar and Pabo, 1993, Science 263:671-673; and PCTPublication No. WO 94/18318.

In a specific embodiment, screening can be carried out by contacting thelibrary members with a Fhit protein (or nucleic acid or derivative)immobilized on a solid phase and harvesting those library members thatbind to the protein (or nucleic acid or derivative). Examples of suchscreening methods, termed “panning” techniques are described by way ofexample in Parmley and Smith, 1988, Gene 73:305-318; Fowlkes et al.,1992, BioTechniques 13:422-427; PCT Publication No. WO 94/18318; and inreferences cited hereinabove.

In another embodiment, the two-hybrid system for selecting interactingproteins in yeast (Fields and Song, 1989, Nature 340:245-246; Chien etal., 1991, Proc. Natl. Acad. Sci. USA 88:9578-9582) can be used toidentify molecules that specifically bind to a Fhit protein orderivative.

5.13. Animal Models

The invention also provides animal models.

In one embodiment, animal models for diseases and disorders involvingcell overproliferation (e.g., as described in Section 5.8.1) areprovided. Such an animal can be initially produced by promotinghomologous recombination between a FHIT gene in its chromosome and anexogenous FHIT gene that has been rendered biologically inactive(preferably by insertion of a heterologous sequence, e.g., an antibioticresistance gene). In a preferred aspect, this homologous recombinationis carried out by transforming embryo-derived stem (ES) cells with avector containing the insertionally inactivated FHIT gene, such thathomologous recombination occurs, followed by injecting the ES cells intoa blastocyst, and implanting the blastocyst into a foster mother,followed by the birth of the chimeric animal (“knockout animal”) inwhich a FHIT gene has been inactivated (see Capecchi, 1989, Science244:1288-1292). The chimeric animal can be bred to produce additionalknockout animals. Such animals can be mice, hamsters, sheep, pigs,cattle, etc., and are preferably non-human mammals. In a specificembodiment, a knockout mouse is produced.

Such knockout animals are expected to develop or be predisposed todeveloping diseases or disorders involving cell overproliferation (e.g.,malignancy) and thus can have use as animal models of such diseases anddisorders, e.g., to screen for or test molecules (e.g., potentialanti-cancer therapeutics) for the ability to inhibit overproliferation(e.g., tumor formation) and thus treat or prevent such diseases ordisorders.

In a different embodiment of the invention, transgenic animals that haveincorporated and express a dominant-negative mutant FHIT gene have useas animal models of diseases and disorders involving celloverproliferation. Such animals can be used to screen for or testmolecules for the ability to specifically inhibit the dominant-negativemutant and thus treat or prevent such diseases and disorders.

6. THE HUMAN FHIT GENE, SPANNING THE CHROMOSOME 3p14.2 FRAGILE SITE ANDRENAL CARCINOMA ASSOCIATED TRANSLOCATION BREAKPOINT, IS ABNORMAL INDIGESTIVE TRACT CANCERS

As described herein, we have isolated and characterized a human geneinvolved in esophageal, gastric, colon, kidney, and other cancers. A200-300 kilobase (kb) region of chromosome 3p14.2, including the fragilesite locus, FRA3B, is involved in homozygous deletions in multipletumor-derived cell lines and in hemizygous deletions in esophageal,gastric, colon, kidney and other cancers. Exon amplification from acosmid contig covering this 200-300 kilobase region allowedidentification of the human FHIT gene, a member of the zinc-bindinghistidine triad gene family, which encodes a ubiquitous 1.1 kilobasetranscript and a 16.8 kDa protein with homology to a protein kinase Cinhibitor gene, another member of the HIT family.

The FHIT locus is composed of 10 small exons distributed over at least500 kilobases, with the three 5′ most untranslated exons mappingcentromeric to the clear cell renal carcinoma associated 3p14.2translocation breakpoint; the remaining exons map telomeric to thistranslocation breakpoint with exon 5, the first amino acid coding exon,falling within the homozygously deleted fragile region, FRA3B, and exons6-10 mapping telomeric to the tumor cell common deleted region and theFRA3B region. Aberrant transcripts of the PHIT locus were found inapproximately 50% of esophageal, stomach and colon carcinomas, and thefamilial t(3;8) renal carcinomas have lost one FHIT allele due todisruption by the translocation.

The aberrant FHIT transcripts usually resulted from abnormal splicing,which often deleted exon 5 or 8, resulting in transcripts which couldnot encode Fhit protein. Thus, chromosome abnormalities at 3p14.2 andFRA3B, resulting in loss of the Fhit protein, are involved in initiationand/or progression of several important types of human cancer.

6.1. Results

The Cosmid Contig

From the 648D4 cosmid library, clones were selected initially using theBE758-6, A6URA, A3, and 1300E3, probes, which were distributed acrossthe homozygously deleted region as shown in FIG. 1A. Cosmid end-cloneswere then isolated and used for the next round of cosmid screening. Thecosmid map was assembled by PCR-amplification of the starting STSs (DNAsequence tags) and new ones developed from cosmid ends, using cosmid DNAtemplates. Additionally, each new STS was tested against the YAC contig(also shown in FIG. 1A), against cell lines with homozygous deletionsand rodent-human hybrids retaining portions of chromosome 3 (LaForgia etal., 1993, Cancer Res. 53:3118-3124; Druck et al., 1995, Cancer Res.55:5348-5355; Bullrich et al., 1995, Cytogenet. Cell Genet. 70:250-254).Six cosmids were assembled into a contig which covered the homozygouslydeleted region.

To define more precisely the homozygously deleted region, which we willrefer to as the fragile region, 42 STS markers, spanning the chromosomalregion from the PTPRG locus to D3S1234, derived from cosmid walking andexon trapping, were tested by PCR-amplification for presence in elevencancer-derived cell lines which had been tested previously with a subsetof markers (data not shown; and Lisitsyn et al., 1995, Proc. Natl. Acad.Sci. USA 92:151-155).

Colon carcinoma-derived LoVo, HT29 and SW480 and gastriccarcinoma-derived AGS cell lines showed similar large deletions such asdepicted by the dotted portion of the top line in FIG. 1A. Coloncarcinoma-derived LS180 and breast carcinoma-derived MDA-MB436 cellsexhibited discontinuous deletions, covering this same region, with mostmarkers lost but some retained. The gastric carcinoma-derived KatoIIIcells appeared to have lost the D3S1481 marker and the telomeric portionof the fragile region, from AP4/5 to D3S2977 (see FIG. 1A). The HKIcells, derived from a nasopharyngeal carcinoma (NPC), had lost theregion between D3S1481 and the AP4/5 marker, while CNE2, anotherNPC-derived cell line had a discontinuous deletion which included aregion near the t(3;8) and the region between D3S1481 and D3S2977. HeLacells also exhibited discontinuous deletions with one deleted regionnear the t(3;8) and between D3S1481 and AP4/5. The NPC-derived CNE1cells were tested with most markers without detection of a deletion.Thus, there are many different tumor associated 3p14.2 chromosomebreakpoints surrounding the t(3;8), the FRA3B locus and the homozygouslydeleted region covered by the cosmid contig.

Isolation of cDNAs

The six cosmids covering the homozygous deletion, shown in FIG. 1A, wereused in exon trapping experiments aimed at identifying genes within thedeleted region. Putative trapped exons were sequenced and sequencesanalyzed using GRAIL 2 of the ORNL GRAIL server. Several trapped exonswere recognized as exons by Grail 2 and were used as probes on northernblots of poly A⁺ RNA from a spectrum of human tissues. Additionally,sequences of trapped exons were compared against nucleotide sequencedatabases. One exon, trapped from a cosmid 76 subclone (c76, FIG. 1A)matched a number of cDNA sequences from breast (Genbank accession#R53187 and #R86313) and fetal liver and spleen (#R1.1128) librariessubmitted by the Washington University-Merck EST Project. A 23 basepair(bp) oligonucleotide primer designed from this sequence (FIG. 2A, primerX8) was used in primer extension to obtain a 5′ extended product of thecDNA by a RACE (Rapid amplification of cDNA ends) reaction (Marathon™cDNA amplification kit, Clontech). The longest product (370 bp) from theRACE reaction detected a ubiquitously expressed 1.1-kb mRNA by northernblot analysis of mRNAs from various normal tissues. The size was similarto the length of the largest cDNA clone isolated from a normal coloncDNA library using the same DNA fragment as a probe. The DNA sequenceanalysis of this full length clone (FIG. 2A) revealed a long 5′untranslated region of more than 350 bp followed by an initialmethionine codon and surrounding sequence which fitted Kozak's rule, anopen reading frame (ORF) of 147 amino acids, a 31 untranslated region, apolyadenylation consensus sequence and a poly A tail. Exon sizes variedwidely, e.g., exon 5 having 120 nucleotides, exon 6 having 146nucleotides, and exon 7 having 30 nucleotides. With reference to FIG.2A, exon 1 consists of nucleotide numbers −362 to −213; exon 2 consistsof nucleotide numbers −212 to −164; exon 3 consists of nucleotidenumbers −163 to −111; exon 4 consists of nucleotide numbers −110 to −18;exon 5 consists of nucleotide numbers −17 to 103; exon 6 consists ofnucleotide numbers 104 to 249; exon 7 consists of nucleotide numbers 250to 279; exon 8 consists of nucleotide numbers 280 to 348; exon 9consists of nucleotide numbers 349 to 449; and exon 10 consists ofnucleotide numbers 450 to 733.

A hydrophilicity plot for the Fhit protein was carried out and is shownin FIG. 6.

This FHIT cDNA, as well as the matching sequences from the EST database,were translated and open reading frame (ORF) amino acid sequences (FIG.2A) compared to the protein databases. The longest EST in the 5′direction was R50713 (which contained sequence found in the 3′ end ofFHIT exon 7, exon 8, and exon 9). The longest EST in the 3′ directionwas R11128 (which contained sequence found in half of exon 2, and inexons 3-6). EST R53187 had the longest span of sequences correspondingto the FHIT cDNA, including 297 nucleotides identical to the FHIT cDNAsequence from exon 2 through a portion of exon 5. Among the best matchesin the database retrieved by computer searches, this 297 nucleotidesequence was the longest stretch of identity with the FHIT cDNAsequence. The next longest stretch of identity was found in EST 11128,with 287 nucleotides identical to the PHIT cDNA sequence starting withinexon 2 until 3 bases before the end of exon 6. A printout of the R50713nucleotide sequence aligned with the FHIT cDNA sequence (cDNA 7F1) andthe R11128 nucleotide sequence is shown in FIGS. 7A-7C. As will benoted, neither of the R53187 nor R11128 nucleotide sequences, or any ofnumerous other EST sequences, span the full FHIT protein coding region.Also, the translations of the R50713, and R11128 sequences in all threereading frames, in both orientations, are shown in FIGS. 8 and 9,respectively, and from none of the translated sequences shown in FIG. 8or 9 can the Fhit protein sequence be deduced.

The full length FHIT cDNA probe was then hybridized to northern blotscarrying mRNA from a spectrum of tissues. As shown in FIG. 3A, the cDNAdetected the ubiquitously expressed 1.1-kb transcript.

Relationship of the cDNA to the Genomic Map of the Region

Oligonucleotide primers from the initially trapped exon were used togenerate intron sequences from cosmid 76; these sequences were used inturn to prepare primers and probes to map the exon (E5 in FIG. 1A) onthe cosmids, YACs and DNA from cancer cell lines with deletions, asillustrated in FIG. 1A. Using cDNA as template, oligonucleotide primerpairs bracketing the exons upstream and downstream of exon 5 were thenused to amplify cDNA fragments to serve as probes for mapping the 5′ and3′ flanking exons on the cosmid contig; these probes demonstrated thatthe cDNA sequences 5′ and 3′ of exon 5 were not within the 648D4 cosmidcontig covering the homozygous deletions. Thus, cosmid libraries fromYACs 850A6 and 750F1, which extend centromeric and telomeric to thefragile region deletions, respectively, as shown in FIG. 1A, wereprepared and screened with the 5′ and 3′ cDNA probes flanking exon 5.Cosmids containing the remaining exons were then used to derive intronsequences using cDNA primers, and the structure of the gene determinedas shown in FIG. 1A. The cDNA consisted of 10 exons which weredistributed among 3 YAC clones (FIG. 1A); exons 1 through 4 mapped toYAC clone 850A6, exon 5 was present in all three YAC clones, and exons 6through 10 mapped to YAC clone 750F1. Only exon 5 fell within the regionof homozygous deletion in tumor-derived cell lines, i.e. within YACclone 648D4. The coding region of the ORF began in exon 5 and ended inexon 9, as shown in detail in FIGS. 2A and 2B.

Most interestingly, the first three exons (E1, E2 and E3) of the genemapped centromeric to the t(3;8) break, between the t(3;8) break and the5′ end of the PTPRG gene, as determined by amplification of these exonsfrom the YAC DNAs and DNAs derived from hybrids carrying portions ofchromosome 3, derived from the t(3;8) break and a FRA3B break (data notshown). Thus, this gene became a strong candidate for involvement ininitiation of the familial RCCs, because one copy of the gene isdisrupted by the translocation.

The homology search in amino acid sequence databases showed asignificant homology to a group of proteins which have a histidine triadmotif, designated HIT proteins (Seraphin, 1992, J. DNA Sequencing &Mapping 3:177-179). The predicted amino acid sequence of the cDNA forthe human gene, designated the Fragile Histidine Triad gene or the FHITgene, is shown in FIG. 4A compared to the other members of the HITfamily. The highest homology of the FHIT protein (˜50% identity) is tothe yeast diadenosine hydrolases (aph1s), shown in FIG. 4A as PAPH1 andCAPH1, identified in S. pombe and S. cerevisiae, respectively (Huang etal., 1995, Biochem. J. 312:925-932). An alignment of the yeast (S.pombe) Ap4A hydrolase (PAPH1) sequence (U32615) with FHIT (cDNA 7F1) isshown in FIGS. 10A-10C. There is not extensive homology. When we did acomputer search for homology stretches between the yeast hydrolase andthe FHIT nucleotide sequences, the result was the small region ofnucleotide homology shown in FIG. 10B The consensus sequence for the HITfamily proteins is shown below the amino acid sequences in FIG. 4A.

To recapitulate, the PHIT gene, which may be the human cognate gene forthe yeast Ap₄A hydrolase gene, spans a >500 kbp region which includesthe t(3;8), the FRA3B and a tumor cell-specific commonly deleted region.

Expression of the FHIT Gene

We had placed the BE758-6 locus and microsatellite marker, D3S1300,within the region of common loss in a variety of tumor-derived celllines and our LOH study of gastric and colon tumors detected a highfrequency of allelic deletion, often involving D3S1300, in the regionbetween the t(3;8) and the D3S1234 locus (see FIG. 1A). Now, thelocalization of both the BE758-6 and D3S1300 loci within the FHIT genelocus, close to the first coding exon, exon 5, suggested that the FHITgene was the target of deletion in uncultured tumors, as well astumor-derived cell lines. To begin an analysis of FHIT transcripts intumor-derived cells, mRNAs from tumor-derived cell lines and normaltissues was studied by northern analysis.

Poly A⁺ RNA from normal tissues and a number of NPC, colon and gastrictumor-derived cell lines, with and without apparent deletions in thefragile region, was tested for hybridization to the FHIT cDNA onnorthern blots (FIGS. 3A and B).

A low level of expression of the FHIT gene occurred in all human tissuestested, as shown in FIG. 3A for spleen, thymus, prostate, testis, ovary,small intestine, colon and peripheral blood lymphocytes. The majortranscript was ˜1.1 kb with a longer transcript at ˜5 kb, which wasbarely detectable or undetectable on some blots. Since the 1.1 kbtranscript matches the size of the full-length cDNA, the longertranscript may represent a precursor RNA which is not fully processed.Similar transcripts were seen in mRNA from brain, heart, lung, liver,skeletal muscle, kidney and pancreas, with the putative unprocessed RNAappearing to be more abundant in lung, small intestine and colon on somenorthern blots.

mRNAs from tumor-derived cell lines with known homozygous deletions inthe fragile region exhibited varying levels of FHIT transcripts (FIG.3B), from barely detectable (FIG. 3B, lanes 2-4, KatoIII, HKI and LoVomRNA, respectively) to almost a normal level (lane 8, LS180), relativeto normal small intestine mRNA (lane 1).

Note that the NPC cell lines with (CNE2, HK1; FIG. 3B, lanes 5, 3) andwithout (CNE1; FIG. 3B, lane 6) homozygous deletions we had documentedexpressed barely detectable FHIT mRNA. The NPC-derived cell line, CNE2,exhibited a possible smaller transcript (FIG. 3B, lane 5), whileColo320, a colorectal carcinoma-derived cell line without a deletion,exhibited an apparently normal-sized FHIT transcript (FIG. 3B, lane 7),although it should be noted that size alone does not imply presence of awildtype transcript. The −1.1 kb bands could harbor transcripts with oneor more small exons missing, since several exons are very small, e.g.exon 7, 30 nucleotides, exon 2, 49 nucleotides, exon 3, 53 nucleotides.One conclusion of the northern analysis is that there was no directrelationship between size or abundance of transcript and detection ofhomozygous deletions in specific tumor-derived cell lines, suggestingthat there may be small deletions in some tumor cell lines which havenot been detected with the available markers.

RT-PCR and cDNA Sequence Analysis of Tumor-Derived mRNA

In order to look for abnormalities in FHIT transcripts from deleted andnondeleted tumor cell lines, we reverse-transcribed mRNAs with (dT)₁₇primer, amplified the cDNA with 5′ and 3′ primers and then reamplifiedusing primers inside the original primers (nested PCR), as described inmethods. Positions of the primers are shown in FIG. 2A. The amplifiedproducts were separated on agarose gels and normal-sized and aberrantfragments were cut from the gels and sequenced (examples of aberrantbands are shown for mRNAs of uncultured tumors in FIG. 3C; RT-PCRproducts from the tumor cell lines were very similar). The tumor-derivedcell lines exhibited a pattern of products ranging from only oneapparently normal-sized amplified transcript to numerous aberrant bandswithout a normal-sized band. Some tumor-derived cell lines exhibitedboth an apparently normal-sized and one or more aberrant bands. Thesequencing of the aberrant bands revealed numerous abnormal products,some examples of which are illustrated in FIG. 1B. Colon tumor-derivedCCL235 and CCL234 cell lines did not show deletion of the STS markerstested, but both showed aberrant transcripts, as illustrated, withCCL235 exhibiting a normal-sized product in addition. HT29 and KatoIIIcell lines both showed homozygous deletion, but the KatoIII cell lineexhibited a deletion of the telomeric portion of the homozygouslydeleted region and not the region containing exons 4 and 5, nor theregion of exon 6, exons which are all missing in the aberrant RT-PCRproduct, as illustrated in FIG. 1B. Numerous other tumor-derived celllines also exhibited aberrant RT-PCR products similar to those shownschematically in FIG. 1B (data not shown). Detailed descriptions ofsimilar aberrant products from uncultured tumors (FIG. 3C) are given inTable 2 and FIG. 2B.

Ten cases of uncultured esophagus, nine of stomach and eight of colontumors were analyzed, and aberrant transcripts were observed in 5, 5 and3 cases, respectively (summarized in Tables 2 and 3 and illustrated inFIG. 2B. TABLE 2 Derivation of FHIT RT-PCR Amplified Products and cDNASequences From Uncultured Tumors of Gastric Organs Cases with aberranttranscripts No. of cases Number Origin of Tumors analyzed of casesCodes^(a) Esophagus 10 5 E3*, E12*, E13* E32*, E37* Stomach 9 5 J1*, J3,J4*, J7, J9* Colon 8 3 9625*, 5586*, 9575*^(a)In cases with asterisks (*), normal tissues from the same organswere analyzed and did not exhibit alterations in the coding regionsequences.

TABLE 3 Aberrant Transcripts Observed in Uncultured Tumors¹Tumor-derived Deletion Insertion⁴ Putative protein⁵ transcript²(position³) Size (bp) Homology Effect coded in frame *E3 a 280-348 72 NSEx 8 loss HIT(−) *E12 a 280-348 — — Ex 8 loss HIT(−) — b 122-516 — — FSafter Ex 6 HIT(−) *E13 a −17-249 — — Ex 5 & 6 loss — — b −17-348 — — Ex5-8 loss — E32 — 280-449 — — Ex 8 & 9 loss HIT(−) *E37 a — 72 NS noneintact — b −73-173 — — Ex 5 loss HIT(+) *9625 a 280-348 — — Ex 8 lossHIT(−) — b −17-279 87 Alu Ex 5-7 loss — — c −110-204   — — Ex 4 & 5 lossHIT(+) *5586 a −17-349 135  Alu Ex 5-8 loss — — b −17-279 37 NS Ex 5-7loss — *9575 a 280-348 — — Ex 8 loss HIT(−) — b  60-181 — — FS after Ex5 HIT(−) — c −110-348   — — Ex 4-8 loss — J1 a −110-(−17) — — noneintact — b −17-279 — — Ex 5-7 loss — J3 — −17-279 173  Alu Ex 5-7 lossHIT(+) J4 — −17-457 305  Alu Ex 5-9 loss — *J7 a −110-249   — — Ex 4-6loss — *J9 a 280-348 — — Ex 8 loss HIT(−)¹All the aberrant transcripts which involve the coding sequence of theFHIT gene are shown in FIG. 3B. Alu, Alu repeat, FS, frameshift; NS, nosignificant homology; Ex, exon.²In tumors with asterisks (*), normal transcripts without alteration ofcoding region sequence were also observed.³The positions of the first and last nucleotides of the deletions areshown according to the nucleotide numbers in FIG. 2A.⁴The position of all insertions was downstream of exon 4.⁵Putative protein coded in frame with the Fhit protein is shown: HIT(+),protein with HIT motif; HIT(−), protein without HIT motif; —, no proteinin frame.The sequence analyses of the aberrant cDNAs revealed absence of variousregions between exons 4 and 9 (Table 3 and FIG. 2B), while the RT-PCRand cDNA sequence analyses of normal tissue mRNAs from the same organsdid not exhibit any alterations of the coding region sequence (Table 2,E3, E12, E113, E37, J1, J4, J9, 9625, 5586, 9575). In 8 of 13 cases withaberrant transcripts, normal-sized transcripts were also observed (FIG.3C; E3, E12, E13, E37, 9625, 9575, J7 and J9; E12 and 9575, not shown),while in 5 of 13 cases normal-sized transcripts were not detected (FIG.3C, J3, J4), or were barely detected (FIG. 3C, E32, 5586, J1). In mostof the aberrant transcripts, the beginning and the end of the deletedportions of the transcripts coincided with splice sites (FIG. 2B),suggesting that the cDNA deletions resulted from the loss of genomicregions containing or surrounding the relevant lost FHIT exons. Theaberrant transcripts can be classified into two groups (class I and II,FIG. 2B): class I transcripts lack exon 5, which has the initialmethionine codon of the FHIT ORF, resulting in the loss of the ORF;class II transcripts have an intact initial methionine codon but do notinclude exon 8, except for 9575b, which exhibited a frameshift afterexon 6. Thus, in all the class II transcripts, the wildtype ORF of exon8, the histidine triad containing domain, is not present. Moreover, someof the class II transcripts exhibited loss only of exon 8 (FIG. 2B; E3,E12a, 9625a, 9575a, J9a), suggesting that exon 8 was the target ofdeletion. Since exon 8 encodes the histidine triad motif, it is likelythat neither class I nor class II transcripts, constituting the majorfraction of aberrant transcripts, can encode a fully functional protein.However, there is an in frame methionine (Met) codon in exon 6 (see FIG.2B), and in some cases insertions contribute an in frame Met (notshown); thus, the majority of aberrant transcripts could encode partialproteins with or without the HIT domain as indicated in Table 3.Insertions of various lengths, of DNA not derived from the FHIT gene,were observed in some transcripts; insertions were found only downstreamof exon 4 (Table 3, 5586a, 5586b, 9625, J3, J4). A minor group ofaberrant transcripts retained intact full length ORFs, but were missingexon 4 (Table 3, J1a), or had insertion of 72 bp of DNA sequence in the5′ noncoding region between exon 4 and 5 (Table 3, E37a, and FIG. 1B).It is possible that such insertions affect translation of the ORF.

In order to determine if the wildtype FHIT cDNA and various cDNAsderived from tumor specific transcripts, which retained the entirecoding region, could be translated in vitro, several recombinantplasmids were constructed, each containing a FHIT gene downstream fromthe T7 promoter and lacking the first noncoding exon. The pFHIT1 plasmidcarried an aberrant cDNA, missing exon 4, from the CCL234 colon cancercell line. Plasmid pFHIT2 carried a cDNA from esophageal tumor E37 withan insertion of 72 bp between exon 4 and exon 5. The pFHIT3 plasmidcontained the wildtype FHIT gene lacking exon 1. The constructs wereused for in vitro translation by rabbit reticulocyte lysate. Analysis oftranslation products (FIG. 4B) showed the predicted 16.8 kDa proteintranslated from each cDNA construct.

The FHIT Protein

The protein sequence predicted by the FHIT cDNA is very similar (57/109amino acid identities; 76/109 or 69%, similarities, as calculated by theNCBI BLAST server) to the S. pombe diadenosine 5′,5′″P¹, P⁴tetraphosphate hydrolase, aph1 (Huang et al., 1995, Biochem. J.312:925-932), as shown in the amino acid alignment in FIG. 4A, wherePAPH1 represents the S. pombe sequence.

The S. pombe aph1 enzyme was cloned by purification of the enzyme, aminoacid sequencing of the N-terminus and design of primers to amplify apartial cDNA; the full length genomic and a cDNA of 1.2 kbp were thencloned, sequenced and translated (Huang et al., 1995, Biochem. J.312:925-932). By similar methods, a human hydrolase (APH1) has beencloned, sequenced and translated (Thorne et al., 1995, Biochem. J.311:717-721) and, surprisingly, does not resemble the S. pombe aph1 genenor the FHIT gene. Since higher eukaryotes appear to possess a single16-21 kDa Ap₄A asymmetrical pyrophosphohydrolase (cited in Thorne etal., 1995, Biochem. J. 311:717-721), it is thus not clear if the FHITgene is a human APH1 enzyme, although it may be a human cognate of theS. pombe aph1 enzyme.

The FHIT gene is also very similar to the S. cerevisiae aph1 geneproduct (CAPH1 in FIG. 4A) with 40% identity and 62% similarity in the50 amino acids between 49 and 102 of the FHIT amino acid sequence, andhigher similarity in the HIT domain. The other proteins or hypotheticalproteins in FIG. 4A are all members of this HIT gene family, a family ofproteins present in prokaryotes, yeast and mammals, described bySeraphin, 1992, DNA Sequencing & Mapping 3:177-179. The signaturefeature of the family is the histidine triad (most commonly HVHVH, aminoacids 94-98 of the FHIT protein, FIG. 4A), which for the case of BHIT(FIG. 4A), the bovine inhibitor of protein kinase C (PKCI1) has beenshown to be a zinc-binding site (Pearson et al., 1990, J. Biol. Chem.265:4583-4591; Mozier et al., 1991, FEBS 279:14-18). The Fhit proteinproduct is only 39% similar to the bovine PKCI1 protein over Fhit aminoacids 12-100, as calculated by NCBI BLAST. Thus, the FHIT gene is notlikely to be the human PKCI1 gene. Functions of the other HIT genes arethus far not known. Furthermore, structural features of family membershave not been studied extensively. The PKCI1 protein has a predictedcontent of 23% α helix and 42% β conformation (31% β sheet and 11% βturn) (Pearson et al., 1990, J. Biol. Chem. 265:4583-4591); theconserved region, including the histidine triad and upstream region werepredicted to be mostly random coil alternating with β sheetconformation, with the HIT domain β sheet. This conformation may bepreserved in the Fhit protein. Also, the HIT domain consists of basicand hydrophobic amino acids and might be expected to be buried insidethe protein, as suggested for the PKCI1 protein (Pearson et al., 1990,J. Biol. Chem. 265:4583-4591).

6.2. Discussion

The meaning of fragile sites for cancer has been a subject ofspeculation for years and the near coincidence of the chromosomalposition of the FRA3B and the t(3;8) translocation at 3p14.2 has beenespecially intriguing. The FRA3B is constitutive; that is, aftertreatment of peripheral blood lymphocytes with ˜0.4 μM aphidicolin,which interferes with the action of DNA polymerase α, the characteristicgaps in chromosome region 3p14.2 are observed in ˜70% of metaphases fromall individuals. So the structural basis for the induction of gaps ispresent in all individuals. It is also known that within the 3p14.2band, some of the induced gaps represent chromosome breaks, which occurpossibly at several sites in the chromatin of an ˜200-300 kilobaseregion (Paradee et al., 1995, Genomics 27:358-361). Thus, the sequencesinvolved in gaps and breaks may occur in more than one site within thefragile region. At other fragile sites such as the folate-sensitivefragile sites on X, FRAXA, FRAXE, FRAXF, the structural basis for thegaps seems to be variable lengths of CCG or CGG triplet repeats andimperfect repeats are more stable than perfect repeats (Chung et al.,1993, Nature Genet. 5:254-258); these fragile sites seem to be singlesites of fragility. Perhaps the FRA3B appears to be the most commonfragile site because it actually represents a collection of differentfragile sites in a small chromosomal region. The specific sequencesresponsible for the breaks at FRA3B in hybrid cells have not beendescribed but we have observed that many tumor-derived cell linesexhibit apparent discontinuous homozygous deletions. FIG. 5 diagrams therelationship between the various types of chromosome breaks in 3p14.2and the organization of the FHIT gene relative to the breaks. Note thatin FIG. 5, the chromosome breaks and deletions in the KatoIII gastriccarcinoma-derived cells leave the coding region intact, but we haveobserved only aberrant FHIT transcript in this cell line. Thus,inapparent chromosomal abnormalities must account for the lack of normaltranscription in KatoIII and other tumor cells; one possibility is thattwo FHIT alleles are present in KatoIII with hemizygous alterations inthe portions of the FHIT genes not homozygously deleted. Anotherpossibility is that alteration near an exon affects splicing.Additionally, some cancer-derived cell lines and uncultured tumorsshowed transcripts with alterations to noncoding regions of the FHITtranscript. These transcripts were transcribed and translated into fulllength protein in a coupled system using a reticulocyte lysate fortranslation (FIG. 4B), but perhaps in the tumor cells from which theywere derived, the lack of exon 4 or insertion of new sequences wouldaffect expression of the Fhit protein. Another puzzle, if the FHIT geneacts as a classical suppressor gene with inactivation of both alleles,is the presence of normal-sized transcripts along with aberrant productsin the RT-PCR amplification products of tumor-derived cell lines such asCCL235 (colon), A549 (lung) and HeLa (cervical). It is possible that theaberrant transcripts, which in most cases might encode partial Fhitproteins, could interfere with the function of a normal Fhit protein.The normal-sized products from these cell lines have not yet been fullysequenced so it is possible that they do not, in fact, represent normaltranscripts. A number of the uncultured tumors also exhibited aberrantand normal-sized products, and sequencing showed that some of thesenormal-sized products were indeed wildtype products. In these cases, thenormal transcripts could have derived from admixed normal cells.

We have not yet observed point mutations within the coding region of anyFHIT transcripts, perhaps suggesting that aberrant. FHIT genes usuallyare the result of deletions.

Aphidicolin, which inhibits the action of DNA polymerase α, induces thegaps and breaks observed in the FRA3B region in normal metaphases; thusin the digestive tract tumors and tumor cell lines we have studied, thegenomic deletions resulting in aberrant transcription and loss offunctional Fhit protein, could have been induced by exposure of theseorgans to other agents which interfere with DNA replication, such asnicotine, caffeine, possibly alcohol and other known carcinogens.Interestingly, zinc deficiency is associated with a high frequency ofesophageal tumors in man (Yang, 1980, Cancer Res. 40:2633-2644) and rat(Fong et al., 1978, J. Natl. Cancer Inst. 61:145-150); zinc deficiencymay cause proliferation of the epithelial cells lining the esophagus(Yang et al., 1987, J. Natl. Cancer Inst. 79:1241-1246), so perhaps zincdeficiency mimics loss of the Fhit protein, which may require bound zincfor its function. It is, therefore, interesting that FHIT gene exon 8,carrying the HIT motif, the presumptive zinc binding site, is a targetof deletion in numerous digestive tract tumors.

Whether or not this region of 3p14.2 contains repeated CCG or CGGtriplets is not yet known, but because there are differences between therare, inherited folate-sensitive fragile sites which have beencharacterized, and the common, constitutive, aphidicolin fragile sites,perhaps a different basis for the fragility should be expected. Thusfar, we have noted that there are many Alu repeats in the telomericportion of the fragile region (not shown) and there is a (TAA)₁₅ repeatin this same commonly deleted region for which the number of repeats ishighly variable. Perhaps other triplet repeats of this type exist in theregion. Also in ˜9 kilobase pairs of sequenced portions of the cosmid S8(telomeric portion of the fragile region, see FIG. 1A), several Alurepeats and a LINE element were encountered; the nucleotide content ofthe sequenced region was 57.4% A and T residues, while the FHIT cDNAnucleotide content was 48% A and T. A high A and T content ischaracteristic of some characterized origins of DNA replication,especially in yeast and, in fact, although higher eukaryotic origins ofreplication have not been identified, it has been speculated that Alurepeats may be connected with replication. Another notable feature ofthe FHIT gene itself is that nearly all the exons end with the sequenceAG, the usual sequence for splice acceptor sites. Based on ourobservation of frequent aberrant splicing in this fragile region, it istempting to speculate that the region is especially rich in sequencesresembling splice acceptor sites.

Interestingly, we have previously observed a homozygous deletion inmouse L cells, which involves several N-terminal exons of the murinePtprg gene (Wary et al., 1993, Cancer Res. 53:1498-1502), and Pathak etal. (1995, Cancer Genet. Cytogenet. 83:172-173) have shown thatmouse-colon and mammary tumors as well as melanomas have abnormalitiesin, the proximal region of mouse chromosome 14 where Ptprg (Wary et al.,1993, Cancer Res. 53:1498-1502) and probably Fhit loci map.

Studies of FHIT gene RT-PCR products from RNA of numerous cell linessuggested that PHIT gene abnormalities could be important not only inairway and digestive tract tumors such as nasopharyngeal, esophageal,stomach and colorectal carcinomas, but possibly also in ovarian,cervical and lung tumors, osteosarcoma, and some leukemias; also abladder and breast carcinoma cell line exhibited homozygous deletions inthe fragile region (Lisitsyn et al., 1995, Proc. Natl. Acad. Sci. USA92:151-155; and out data). Thus, uncultured tumors of these types shouldbe tested for FHIT gene abnormalities.

Clear cell RCCs might also be expected to involve FHIT gene aberrationsbecause the FHIT gene is disrupted by the familial RCC translocationbreak in 3p14.2 and the translocation/FRA3B region is the target ofallelic loss in most sporadic clear cell RCCs (Druck et al., 1995,Cancer Res. 55:5348-5355). Since the FHIT ORF is contained in exons 5through 9, translocated to chromosome 8 in the t(3;8) family, it ispossible that both alleles could still be expressed in some or alltissues; we have found a few polymorphisms within the ORF but none yetwhich distinguishes the two allelic FHIT transcripts in the t(3;8)lymphoblastoid cell lines (data not shown). If the FHIT gene disruptionis the first “hit” to a suppressor gene in this family, then the secondFHIT allele should be altered in the t(3;8) tumors. Since we have notyet detected point mutations in the FHIT gene, the best way to look foralterations of the FHIT gene in t(3;8) RCCs would be to amplify the FHITreverse transcript, as done for uncultured tumors in this study. We havedone this experiment for RNA from two RCC cell lines and two unculturedRCCs, all from sporadic tumors, and have observed normal-sized products,which have not yet been cloned and sequenced. Nor have we yet observedhomozygous deletions in RCCs using a subset of STS markers in thefragile region. Nevertheless, it would be surprising if the FHIT gene isnot involved in some sporadic RCCs.

Since the FHIT gene is probably ubiquitously expressed, it may not besurprising if it can serve as a tumor suppressor gene for specifictissues of many different organs, apparently predominantly of thedigestive tract, or maybe predominantly organs with epithelial celllinings. Another common denominator of the types of tumor exhibitingaberrant FHIT alleles might be that they are predominantly organsdirectly exposed to environmental carcinogens; some of the types oftumors exhibiting FHIT gene aberrations occur very frequently inrestricted regions of the globe, NPC in China, gastric cancer insoutheast Asia, and often there are environmental factors at play. Apossible role for EBV in promotion of Chinese NPCs might bethrough-viral DNA integration into the FRA3B region, suggested by theprevious experiments of Rassool et al. (1992, Am. J. Hum. Genet.50:1243-1251), showing apparent preferential integration of exogenousDNA into induced fragile sites in cultured cells. Similarly humanpapillomaviruses associated with cervical carcinomas might promoteinduction of the FRA3B, contributing to the loss of heterozygosity on 3pin uterine cancers (Yokota et al., 1989, Cancer Res. 49:3598-3601), andpossibly to inactivation of the FHIT gene. Perhaps the t(3;8) familymembers, carrying one disrupted FHIT gene succumb to kidney tumorsrather than colon or esophageal tumors due to specific types ofenvironmental agents to which they are exposed.

We observed strong similarity of the FHIT gene to S. pombe and S.cerevisiae Ap₄A hydrolases. Specific roles for the diadenosine, Ap₄A,have not been defined (Huang et al., 1995, Biochem. J. 312:925-932) andit is not clear that the Ap₄A hydrolase activity is the only or even themajor in vivo function of these proteins. Expression of the S. pombeaph1 in S. cerevisiae did not inhibit growth, but for unknown reasonsthe S. pombe enzyme was not expressed at a high level (Huang et al.,1995, Biochem. J. 312:925-932). Very little is known of the function ofthe other members of the HIT family. If indeed the FHIT gene is thecognate of the S. pombe aph1 gene identified as an Ap₄A hydrolase, thenthe strong conservation (69% similarity) between the yeast and humangene suggests important functions. Whether the FHIT gene does or doesnot encode an Ap₄A hydrolase, it is likely that the study of the S.pombe and S. cerevisiae hydrolase knockouts and other types of mutationswill be useful in understanding the functions of the Fhit protein.

There is some suggestion that as an intracellular regulatory molecule,the Ap₄A diadenosine may regulate ability of cells to adapt to metabolicstress such as heat, oxidation, and DNA damage; thus deviation fromabnormal level of Ap₄A may result in inability of cells to adapt toenvironmental stresses imposed by carcinogens or viruses which causegenetic damage.

6.3. Material and Methods

Tissues and Cell Lines

Matched normal and cancerous tissues from patients with primaryesophageal, colon and stomach carcinomas were obtained immediately aftersurgery. Tumors were dissected to eliminate normal tissue beforepreparation of DNA. Many cell lines were obtained from the ATCC. The RCkidney cell lines were kindly provided by E. Lattime.

RNA Extraction and Reverse Transcription

Total and poly A⁺ mRNA was extracted from cell lines and tissues usingthe RNAzol kit (TelTest, Inc., Texas) or the FastTrack Kit (Invitrogen),respectively. To obtain mRNA from tissues, fresh specimens were frozenimmediately after excision, and stored at −85° C. or in liquid nitrogenuntil extraction of mRNA. RNA was stored as a pellet under ethanol orsolubilized in RNAse-free water and kept at −70° C. Reversetranscription was performed in 30 μl final volume of 50 mM tris-HCl pH8.3, 75 mM KCl, 3 mM MgCl₂, 10 mM DTT, 2 μM dNTPs, 500 ng oligo-dT, 600units MMLV-RT (BRL), 40 units RNasin (Promega), and 2 μg RNA. Thisreaction was incubated at 37° C. for 90 min and boiled for 5 min.

DNA Sequence Analysis

cDNA, genomic clones and putative exons were sequenced using primersspecific for vector flanking sequences (T3, T7 etc.) and varioussynthetic oligonucleotides. RT-PCR products were directly sequencedafter isolation of bands from low melt agarose and purification bycolumn chromatography (Qiagen, Chatsworth, Calif.). Sequencing ofdouble-stranded plasmids, PCR products and phage or cosmid genomicclones was performed using Taq DyeDeoxy Terminator Cycle Sequencing Kits(Applied Biosystems, Inc. (ABI)); reaction products were electrophoresedand recorded on the 373 or 377 DNA sequencer (ABI). Sequences wereanalyzed using GCG, BLAST, and GRAIL software.

PCR Amplification

The oligonucleotides for generating probes, PCR products and RT-PCRproducts were designed using the computer program Oligo 4.0 (NationalBiosciences). For Southern blots, probes were produced by PCRamplification using various FHIT specific primers, as indicated inresults. Sequences and positions of some primers are shown in FIG. 2A.

PCR reactions were carried out in 12.5 or 25 μl final volume with 1-100ng of template, 20-40 ng primers, 10 mM tris-HCl pH 8.3, 50 mM KCl, 0.1mg/ml gelatin, 15 mM MgCl₂, 200-600 mM dNTPs and 0.5-2.5 units Taqpolymerase (ABI). The amplifications were performed in a Perkin-ElmerCetus thermal cycler for 30 cycles of 94° C. for 30 s (fordenaturation), 60° C. (varied for specific primer pairs) for 30 s (forannealing), and extending at 72° C. for 30-45 s. The PCR products werevisualized in ethidium bromide stained low melting agarose gels. Thebands of amplified DNA were excised from gels and purified for labelingor sequencing.

DNA Preparation and Southern Blot Hybridization

Cellular DNAs were isolated and Southern blots prepared usingconventional methods. Probes were labeled by random-priming with[³²P]-dCTP (NEN) and hybridized to membranes in 0.75 M NaCl, 50%formamide at 42° C. overnight. Final washes of membranes were in 0.1×SSCand 0.1% SDS at 65° C. for 30 min. Hybridized filters were exposed toXAR-2 X-ray film (Kodak) with intensifying screens for times varyingfrom 1 to 72 h.

Identification of YACs

We and others (Boldog et al., 1993, Proc. Natl. Acad. Sci. USA90:8509-8513; Boldog et al., 1994, Genes Chrom. Cancer 11:216-221; Wilkeet al., 1994, Genomics 22:319-326; Michaelis et al., 1995, Cancer Genet.Cytogenet. 81:1-12; Kastury et al., 1996, Genomics, in press) hadpreviously identified the 850A6 clone from the Genethon mega YAC libraryas containing the D3S1300 and D3S1481 markers (Roche et al., 1994, Hum.Mol. Genet. 3:215). Overlapping YACs were identified by analysis of theGenethon database information.

Identification of Region Specific STSs

A number of STSs were available from our work with the 850A6 YAC. TheA6URA marker was from the 850A6 URA end, A3 was from an Alu-vectoretteamplified fragment of 850A6; BE758-6 and D3S1480 amplified fragmentswere used as probes to select phage genomic clones from which endsequences were obtained and sequence tagged. A phage genomic clone forD3S1300 was selected from the 850A6 phage library and end clone D1300E3isolated. Other D3S and WI marker primer pairs were obtained fromResearch Genetics or were synthesized from sequences provided in the WIdatabase. From sequencing of a phage genomic subclone from the 648D4YAC, a (TAA)₁₅ trinucleotide repeat was found and designated locus ph13;the AP4/5 STS was derived from partial sequencing of a cosmid subcloneof the 648D4 YAC.

Cosmid Mapping

High molecular weight YAC containing yeast DNA in agarose plugs waspartially restricted with the Sau3AI enzyme, and subcloned into a cosmidvector. This cosmid library was initially screened with DNA probesderived from STSs previously mapped to this region. The ends of theinsert DNAs flanking the cosmid vector were sequenced to find new STSs,which were used as probes to rescreen the cosmid libraries.

Exon Trapping and cDNA Cloning

The cosmid DNAs were partially restricted with Sau3AI enzyme, run on a1.0% agarose gel, and fragments larger than 2 kbp cut out and subclonedinto the pSPL vector and transfected into COS-7 cells, according to themanufacturer's instructions. The DNA inserts trapped between the splicesites of the vector were sequenced by a primer supplied with the vector(GIBCO/BRL). The cDNA was extended in the 5′ direction byPCR-amplification of a total human fetal brain cDNA using anexon-specific primer (X8, nucleotide numbers −19 to −41) and a RACEreaction kit (Clontech). The normal colon cDNA library was purchasedfrom Clontech.

FHIT Exon Mapping

The genomic sequences of exon-intron junctions of the FHIT gene weredetermined by sequencing the positive cosmids with primers derived fromthe cDNA. Localization of each exon of the FHIT gene was determined byPCR amplification using primers derived from each exon with YAC andchromosome 3 hybrid DNAs as templates. The primer sequences used toobtain cDNA probes flanking exon 5 were: 5′TCTGCTCTGTCCGGTCACA3′ (SEQ IDNO:70) (nuc. #−355 to −337) with primer X8 (shown in FIG. 2A) for 5′flanking exons; 5′ATGTCCTTGTGTGCCCGCT3′ (SEQ ID NO:71) (nuc. #105 to123) with 3D2 (see FIG. 2A) for 3′ flanking exons.

Northern Blot and Hybridization

Two μg of mRNAs were electrophoresed through 1.5% agarose gel in 2.2 Mformaldehyde and 1×MOPS buffer and blotted to a positively chargedmembrane by standard procedures. Northern blot filters of multiplenormal tissue mRNAs were purchased (Clontech, Palo Alto, Calif.). TheFHIT cDNA probe for hybridization was obtained using the FHIT cDNA astemplate for PCR-amplification with the following primer pair:5′TGAGGACATGTCGTTCAGATTTGG3′ (SEQ ID NO:72), nuc. #-7 to 17; and5′CTGTGTCACTGAAAGTAGACC3′ (SEQ ID NO:73), nuc. #449 to 429. Probes werelabeled by random-priming with [³²P]-dCTP and 2×10⁶ cpm/ml washybridized to each filter. Hybridizations were at 42° C. for 16 hours inSSPE buffer (5×SSPE, 10× Denhardt's solution, 0.1 mg/ml carrier DNA, 50%formamide, 2% SDS). Final washes were in 0.1×SSC, 0.1% SDS at 50° C. for30 min before exposure at −80° C. to XAR-2 films (Kodak) withintensifying screens on both sides.

Nested RT-PCR and Sequencing of cDNAs

First strand cDNAs were synthesized and 1 μl of each product wassubjected to a first round of PCR amplification with 30 cycles of 95° C.for 20 sec, 60° C. for 30 sec, and 72° C. for 1 min with 5%dimethylsulfoxide and 0.5 mM spermidine in 10 μl reaction volume understandard conditions, using primers 5U2 and 3D2, indicated in FIG. 2A.One μl of the reaction products, after 20-fold dilution, was subjectedto a second round of PCR amplification using nested primers 5U1 and 3D1(shown in FIG. 2A), under the conditions noted above, except thereaction volume was 30 μl. The PCR products were run, on 1.5% agarosegels, stained with ethidium bromide, purified and 2.5 ng sequenced usingthe 5U1 primer.

In Vitro Transcription and Translation

Three different fragments of DNA, containing the FHIT gene were obtainedby PCR, using oligonucleotides UR5 (5′CTGTAAAGGTCCGTAGTG3′ (SEQ IDNO:74), nuc. #-171 to −154 in FIG. 2A) and O6 (5′CTGTGTCACTGAAAGTAGACC3′(SEQ ID NO:75), the reverse complement of nuc. #429-449). Amplificationswere performed in 100 μl final volume of 10 mM Tris-HCI (pH 8.9), 50 mMKCl, 1.5 mM MgCl₂, 200 μM deoxynucleotide triphosphates, 10 ng RT-PCRproducts and 2.5 U Taq polymerase using an Omni Gene Thermal Cycler. 25PCR cycles consisted of 94° C. 1 min, 52° C. 1 min and an extension stepat 72° C. 45 sec. PCR products were separately inserted in a PCRIIplasmid using the TA cloning system (Invitrogen). Recombinant vectors,containing the normal FHIT and aberrant genes under the control of theT7 promoter, were sequenced and used for in vitro transcription andtranslation.

The in vitro transcription and translation reactions were performed byTnT Coupled reticulocyte systems (Promega) in a final volume of 50 μlcontaining ½ volume rabbit reticulocyte lysate, 1 μg recombinant plasmidDNA, 10 U T7 polymerase, 20 μM amino acid mixture without methionine, 40μM ³⁵S-methionine (Amersham) and 40 U RNasin ribonuclease inhibitor.Reactions were carried out for 90 min at 30° C. The synthesized proteinswere analyzed by SDS polyacrylamide gel electrophoresis (SDS-PAGE) andautoradiography.

7. THE FHIT GENE AT 3p14.2 IS ABNORMAL IN LUNG CANCER

The FHIT gene is disrupted by the t(3;8) chromosomal translocationobserved in a family with renal cell carcinoma and contains the FRA3Bfragile site and the target of homozygous deletions in various humancancer derived cell lines. The study in Section 6 hereof indicates thatFHIT gene abnormalities often occur in primary digestive tract cancers.

Deletions of the short arm of chromosome 3 occur at a very highfrequency and in early phases of lung carcinogenesis suggesting thatthis chromosomal region contains crucial genes for lung cancerdevelopment. We isolated the FHIT gene, located at the chromosomal band3p14.2, and found that it contains the FRA3B fragile site. The gene isdisrupted in the t(3;8) translocation observed in a family with renalcell carcinoma and resides in a region which shows allelic losses invarious human malignancies. In this study in Section 7 hereof, we haveanalyzed the role of the FHIT gene in cancers associated withcarcinogenic exposure, that is lung cancer of the small-cell andnon-small-cell type. Analysis of 59 tumors and paired normal lungtissues was performed by reverse transcription of FHIT transcriptsfollowed by PCR amplification and sequencing of products; allelic lossesaffecting the gene were evaluated by microsatellite polymorphismsanalysis. About 80% of small-cell lung tumors and 40% of non small-celllung cancers showed abnormalities in RNA transcripts of FHIT and 88% ofthe tumors exhibited loss of FHIT alleles. Abnormal lung tumortranscripts lack two or more exons of the FHIT gene. All the casesshowing abnormal transcripts also had loss of one allele of the FHITgene. The results indicate abnormalities of this gene in nearly all SCLCand in a high proportion of NSCLC, suggesting a critical role for theFHIT gene in lung carcinogenesis.

7.1. Results

RT-PCR and cDNA Sequence Analysis

Small Cell Lung Cancer (SCLC)

In order to study abnormalities in FHIT transcripts from tumors andnormal tissues, we reverse-transcribed mRNAs and amplified the cDNAs bynested-PCR as described in methods. Fourteen primary tumor samples andone cell line (83L) were studied. In two cases matched normal lungparenchyma was also analyzed. Eleven of the 14 cases (79%) analyzed byRT-PCR showed the presence of abnormal transcripts (FIG. 11). Theanalysis of the amplified transcripts from the primary tumorsconsistently revealed the presence of two abnormal bands of ˜360 bp(type I) and ˜250 bp (type II). Seven cases displayed both type I and IIabnormal transcripts whereas four cases showed only the type I band. Intwo samples (FIG. 11A, case 107; FIG. 12A, case 45) as well as in thetumor-derived cell line (FIG. 12A, case 83L) the normal sized transcriptwas undetectable while in the other nine cases a normal-sized band ofvarying intensity was observed.

In one patient (FIG. 12A, case 83) we examined the primary tumor, atumor derived-cell line and a normal lung specimen. Whereas in thenormal lung only the normal transcript was detected, the primary tumorexhibited the type I and II abnormal transcripts together with a normalsized transcript and the tumor-derived cell line displayed the type Iabnormal transcript and a novel band of −420 bp (FIG. 12), probablygenerated following in vitro subculturing. Accordingly, cytogeneticanalysis of this cell line revealed extensive chromosomal instabilityresulting in the presence of dicentric and tricentric chromosomes,telomeric associations and double minutes. FISH analysis with a paintingprobe of chromosome 3 showed the occurrence of several structuralrearrangements of this chromosome including a translocation of the 3parm with a breakpoint in 3p14-21 (data not shown). Interestingly, in thecell line, the normal-sized transcript was undetectable suggesting thatthe normal-sized product observed in the primary tumor reflected thepresence of normal cells infiltrating the tumor specimen.

Abnormal and normal-sized bands were separated on agarose gel, cut andsequenced (FIG. 12B). Sequence analysis of the aberrant bands revealedthat the type I transcript corresponded to absence of exons 4 to 6 (nt−111 to 249) of the FHIT cDNA sequence (to be accorded GenBank accession# u46922), resulting in a junction between exons 3 and 7 of the FHITcDNA. A loss of exons 4 to 8 (nt −111 to 348), resulting in fusion ofexons 3 and 9 was found in the type II transcript. In each aberranttranscript the fusion junctions coincided with splice sites.

Sequence analysis of the normal-sized bands revealed that they containedthe normal FHIT cDNA, possibly reflecting the contribution of normalcells infiltrating the tumor specimens.

Since both types of aberrant transcripts lacked exon 5, containing theinitial methionine codon of the FHIT open reading frame (see Section 6),and type II transcripts also lacked nearly the entire coding region,including exon 8, containing the highly conserved HIT motif (see Section6), it is likely that the results of these aberrant fusion transcriptswould lead to loss of FHIT function.

Sequencing of the normal-sized RT-PCR product amplified from normaltissues (cases 45 and 83) revealed presence of the wildtype FHITtranscript.

Non Small Cell Lung Cancer (NSCLC)

RNA from 45 primary NSCLC tumors and matching normal lung parenchymasamples were similarly studied; 18 of 45 tumors (40%) displayed aberrantRT-PCR products. A detailed description of these results is summarizedin Table 4. TABLE 4 RT-PCR, SEQUENCING AND LOH RESULTS IN NSCLCCASE/TYPE RT-PCR* SEQUENCE LOH°  1_(ADC) N Normal YES A del ex3→7 (nt−111→4249)  2_(SQC) N Normal NE A del ex4→8 + (nt −17→279) A del ex4→9(nt −17→348)  3^(Δ) _(MUCOEp) N Normal NE A del ex3→8 (nt −111→279) 4_(SQC) N Normal YES A del ex4→8 (nt −17→279)  5_(ADC) N Normal YES Adel ex3→9 (nt −111→348)  6_(ADC) N Normal YES A del ex3→8 (nt −111→279) 7_(ADC) N Normal YES A del ex3→9 (nt −111→348)  8_(SQC) N Normal YES Adel ex3→7 (nt −111-249)  9_(SQC) N Normal NE A del ex3→9 (nt −111→348)10_(SQC) N NE YES A 11_(ADC) N NE YES A 12_(ADC) N NE YES A 13_(SQC) NNE YES A 14_(ADC) N Normal YES A del ex3→9 (nt −111→348) 15_(ADC) N NENI A del ex3→9 (nt −111→348) 16_(ADC) N NE NE A A 17_(SQC) N NE A delex4→8 (nt −17→279) 18_(ADC) N NE NE AN = Normal TranscriptA = Abnormal Transcript^(Δ)In Normal Lung Parenchyma: del ex3→9°Loci analyzed: D3S4103, (ph13), D3S1234, D3S1313, D3S1312NE: Not EvaluatedNI: Non Informative

The RT-PCR amplified products from the transcripts present in thesetumors consisted of one or two abnormal bands, always accompanied by anormal-sized transcript (FIG. 13A). All the paired normal lung RNAs fromthe same lung cancer cases showed the presence of the normal FHITproduct only, except one case (case 3 in Table 4) which displayed anaberrant product differing in size from the aberrant product observed inthe corresponding tumor. Sequence analysis of the aberrant fragmentsrevealed a range of RT-PCR products with losses of various exons from 4to 9 (Table 4 and FIG. 13B), including an RT-PCR product-missing exons 4to 8, resulting in a junction of exons 3 and 9 (nt −111 to 348),products missing exons 4 to 6 or 7, fusing exon 3 to exon 7 or 8,respectively, and products missing exon 5 to exon 7 or 8, resulting injunctions between exon 4 and 8 or exons 4 and 9, respectively. All thetypes of abnormal transcripts observed lacked exon 5, the first codingexon, and half (6/12) of the abnormal transcripts characterized bysequence analysis also showed loss of exon 8 containing the HIT domain.Sequence analysis of the normal-sized transcript amplified from RNA ofthe normal tissue of these patients revealed a normal FHIT cDNAsequence. A small alternatively spliced region at the beginning of exon10 from nucleotides 450 to 460, outside the open reading frame of theFHIT gene, was observed in the normal-sized transcript present in thetumor and in the corresponding normal tissue of several patients.

Loss of Heterozygosity (LOH) Analysis

To look for allelic losses in tumor samples, a PCR-based approach wasused using primers which amplify polymorphic microsatellite markersinternal and flanking the FHIT gene. DNA from tumor and correspondingnormal tissues from 28 NSCLC and 7 SCLC cases were analyzed for alleliclosses at locus D3S4103 (ph 13) (see Section 6), internal to the FHITgene, and at loci located in flanking regions, centromeric (D3S1312 at3p14.2) (Druck et al., 1995, Cancer Res. 55:5348-5353) and telomeric(D3S1234 at 3p14.2 and D3S1313 at 3p14.3) to the FHIT gene. (See Table5). TABLE 5 LOH Frequency at 3p14.2 Loci in SCLC and NSCLC D3S1234D3S4103 D3S1212 20/25 (80%) 20/25 (80%) 12/15 (80%)

In NSCLC samples LOH at loci D3S4103 and D3S1234 was found in 17 of the21 informative cases (81%). The combined frequency of losses affectingthese two loci was 88% (23/26 of the informative cases). Ten of 13 (76%)and 9 of 11 (81%) tumors also showed LOH at D3S1312 and at D3S1313 loci,respectively, indicating a large deletion in this genomic region. InSCLC cases, 3 of 4 informative patients showed LOH at D3S4103 andD3S1234, and 4 of 5 informative cases had lost at least one of theseloci. Overall 12 of 12 (100%) of the informative tumors which exhibitedabnormal FHIT transcripts, showed allelic losses at one or more of theloci tested.

7.2. Discussion

Three distinct chromosomal regions of 3p, which include 3p25,3p21.3-p21.2 and 3p12-p14.1, are believed to harbour gene(s) involved inlung cancer on the basis of the high frequency of allelic loss inprimary tumors and defined homozygous deletions in lung cancer derivedcell lines; extensive efforts have been made to define small commonregion of loss in order to isolate tumor suppressor genes in thesechromosomal regions.

Deletions of 3p constitute particularly useful genetic markers sinceseveral studies have reported that they occur in the early stages oflung carcinogenesis, such as bronchial dysplasia and metaplasia(Sundaresan et al., 1992, Oncogene 7:1989-1997; Sozzi et al., 1991,Cancer Res. 51:400-404; Hung et al., 1995, JAMA 273:558-563). Moreoverallelic loss on chromosome 3p in primary SCLC has been suggested torepresent an unfavorable prognostic factor (Horio et al., 1993, CancerRes. 53:1-4).

The study disclosed herein describes the occurrence of abnormalities intranscripts of the FHIT gene, located at 3p14.2, in at least 80% of SCLCand 40% of NSCLC with 88% of the cases also exhibiting loss of one FHITallele. Since the RT-PCR nested amplification would detect only internalalterations in the FHIT gene transcripts, this is a conservativeestimate of the involvement of the FHIT gene in SCLC and NSCLC.

The lung tumor transcripts were missing two or more exons of the FHITgene. While in NSCLCs a varying pattern of abnormal transcripts wasdetected, in SCLCs the amplified transcripts were either missing exons 4to 6 or exons 4 to 8. Both types result in loss of exon 5, containingthe initial methionine codon (see Section 6), with the second type alsoshowing loss of exon 8 containing the highly conserved HIT domain (seeSection 6). The consequence of the loss of these exons is that noin-frame Fhit protein could be produced.

Two cases of primary SCLC and a tumor cell line lacked the normal-sizedtranscript, which was also underrepresented in the remaining cases. Inaddition, one primary tumor exhibited two abnormal transcripts and anormal transcript, while a normal-sized product was not amplified fromthe cell line established from this tumor. These observations suggestthat in the SCLC RNA the wild-type transcript could have derived fromadmixed normal cells.

In RNAs from NSCLCs the abnormal RT-PCR amplified products weresometimes less abundant than the normal-sized RT-PCR amplified products.A possible explanation could be the heterogeneous, often multifocalnature of these neoplasms, which arise as a consequence of the chronicexposure of the entire bronchial “field” to carcinogens, resulting inthe presence of different cell clones carrying different genetic changes(Kim et al., 1993, Am. J. Pathol. 142:307-317; Barsky et al., 1994, Mod.Pathol. 7:633-640; Ebina et al., 1994, Cancer Res. 54:2496-2503). Inaddition, the tumor samples contained variable amounts of normal stromaltissue (stromal infiltration is known to occur in non small-cell tumors)(Rabbitts et al., 1989, Genes Chrom. Cancer 1:95-105). The completeallele loss seen in the SCLC cell line and in several SCLC primaryspecimens and lack of complete loss of alleles in NSCLC supports thisinterpretation. It is also possible that the abnormal transcripts areless stable then the wild type product.

In the corresponding normal tissue of the patients showing abnormaltumor transcripts we have observed a normal FHIT product by PCR andsequence analysis.

Of particular interest was one NSCLC patient (case 3 of Table 4) with amucoepidermoid carcinoma of the lung who subsequently developed a renalcell carcinoma. In the normal lung parenchyma of this patient anabnormal transcript missing exons 4 to 8 was detected. This findingraises the possibility that a constitutional alteration within the FHITgene could be associated with a predisposition to develop both lung andrenal cancer or other types of multiple primary tumors. However thisalteration could have been somatically acquired because carcinogenexposure can induce transformation in several fields of the bronchialepithelium through the induction of different genetic changes which arealso detectable in early preneoplastic lesions.

The high frequency (88%) of loss of one FHIT allele observed in lungtumors of both small cell and non small cell type is noteworthy.Although we did not determine a minimal region of loss in our cases,these findings support the idea that inactivation of the FHIT gene couldhave occurred by a mechanism of loss of one allele and alteredexpression of the remaining one.

This model is consistent with the observation that the FHIT gene spans acommon fragile region, FRA3B, where abnormalities such as deletionscould be more frequent than point mutations. Tumors associated withcarcinogen exposure, such as cancers of the aerodigestive tract, couldbe particularly susceptible to alterations of the FHIT gene. Due to itsetiology, lung cancer is the likely to be strongly and directlyassociated with the effects of agents which interfere with DNAreplication, such as nicotine and mutagens like benzo(a)pyrene containedin cigarette smoke. Breakage in a fragile site containing gene as aconsequence of physical, chemical and biological agents can thus beexpected. Expressivity of the FRA3B fragile site in peripheral bloodlymphocytes of patients with cancer has been investigated; theexpression of FRA3B appeared to be influenced by habitual tobaccosmoking and significantly higher expression was reported in lung cancerpatients (Murata et al., 1992, Jpn. J. Hum. Genet. 37:205-213).

High levels of intracellular diadenosine 5′,5′″-P1,P4 tetraphosphate(Ap4A) have been detected at the G1-S boundary (Weinmann-Dorsch et al.,1984, Eur. J. Biochem. 138:179-185) and a role for Ap4A in thestimulation of DNA polymerase activity has been proposed (Baxi et al.,1994, Biochemistry 33:14601-14607). It seems plausible that loss offunction of the FHIT gene could result in the constitutive accumulationof AP4A and in the stimulation of DNA synthesis and proliferation. Thusloss of FHIT function could initiate the malignant process bystimulating the proliferation of the cells that are the precursors ofdigestive tract cancer and lung cancer.

7.3. Experimental Procedures

Tumors

The 59 tumors, including 25 cases of adenocarcinomas, 19 squamous cellcarcinomas, 1 mucoepidermoid carcinoma and 14 small cell lungcarcinomas, were obtained from surgically resected lung cancer patientsat Istituto Nazionale Tumori (Milano, Italy). A cell line (83L) wasestablished from one small-cell tumor (83T). Twenty-nine NSCLCs were instage I, 9 in stage II and 6 in stage III. The tumors were classifiedhistologically according to the Histological Typing of Lung Tumors bythe World Health Organization (1987) and staged according to the TNMclassification of malignant tumors defined by the International UnionAgainst Cancer (1987). Most cases (54 out of 59) were from male patientsand the mean age of cases at presentation was 63 years. Matched normallung parenchyma tissue samples were taken at a most distant site fromthe tumor or in a different segment or lobe, as a source for the normalRNA and DNA.

RNA Extraction and Reverse Transcription

Tumor and normal specimens were frozen immediately after surgicalresection and stored at −80° C. Total mRNA was extracted from frozentumor and normal lung tissues using guanidinium-LiCl separation(Sambrook et al., 1989, Molecular cloning: A Laboratory Manual. ColdSpring Harbor Lab. Press, Plainview, N.Y.) or the RNA-STAT kit (TelTEST, Inc., Texas). cDNA was synthesized from 1 μg of total RNA. Reversetranscription was performed in a 20 μl volume of 1× first strand buffer(GIBCO), 10 mM DTT (GIBCO), 500 μM dNTPs, 50′ ng/μl oligo-dT, 0.3 μg/μlrandom primers, 16.5 U RNAsin (PROMEGA), 300 U Superscript II (GIBCO).The samples were first denatured for 5 min at 95° C. and incubated at37° C. for 60 min. The reaction was stopped by inactivating the enzymeat 94° C. for 5 min. The reaction was diluted to 30 μl and 1 μl was usedfor subsequent PCR amplification.

RT-PCR and cDNA Sequencing

1 μl of cDNA was used for a first PCR amplification performed in avolume of 25 μl containing 0.8 μM of primers 5U2 and 3D2 (see Section6), 50 μM of each dNTP (TAKARA), 1×PCR buffer and 1.25 U Taq Polymerase(TAKARA). The PCR reaction consisted of an initial denaturation at 95°for 3 min and 25 cycles of 15 sec at 94°, 30 sec at 62°, 45 sec at 72°and a final extension of 5 min at 72°, using a Perkin Elmer PCRThermocycler. The amplified product was diluted 20-fold in TE buffer and1 μl of the diluted reaction product was subjected to a second round ofPCR amplification using nested primers 5U1 and 3D1 (see Section 6) for30 cycles under the above conditions. The PCR products were resolved on1.5% ethidium-bromide stained Metaphor gel (FMC). Bands were cut fromgels and DNA was purified using a QIA quick gel extraction Kit (QIAGEN).5-50 ng of cDNA, depending on the size of the PCR products, weresequenced using primers 5U1 and 3D1 by the dideoxynucleotide terminationreaction chemistry for sequence analysis on the Applied BiosystemsModels 373A and 377 DNA sequencing systems.

LOH Analysis

DNAs from frozen tumor and normal tissues were extracted using standardmethods (Sambrook et al., 1989, Molecular cloning: A Laboratory Manual.Cold Spring Harbor Lab. Press, Plainview, N.Y.). Analysis of alleliclosses was performed using a PCR-based approach. Primers which amplifypolymorphic microsatellite markers were used for the following loci:D3S4103 (ph13) (3p14.2) internal to the FHIT gene (see Section 6),D3S1234 (3p14.2), D3S1313 (3p14.3) and D3S1312 (3p14.2) flanking thegene. The sequence of all oligonucleotide primers will be availablethrough the Genome Data Base. Twenty cycles of amplification werecarried out at 55°-60° C. annealing temperature as appropriate for eachprimer.

For informative cases, allelic loss was scored if the autoradiographicsignal of one allele was approximately 50% reduced in the tumor DNA,compared to the corresponding normal allele. The loci displayingmicrosatellite instability were not scored for allelic loss.

8. Deposit of Microorganisms

E. coli strain DH5α carrying plasmid p7F1, containing a full-length FHITcDNA as a BamHI-XbaI insert into the pBluescript SK⁺ vector (Stratagene)was deposited on Jan. 30, 1996, with the American Type CultureCollection, 1201 Parklawn Drive, Rockville, Md. 20852, under theprovisions of the Budapest Treaty on the International Recognition ofthe Deposit of Microorganisms for the Purposes of Patent Procedures, andassigned accession number 69977.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theappended claims.

Various publications are cited herein, the disclosures of which areincorporated by reference in their entireties.

1-21. (canceled)
 22. An isolated antibody that binds to at least onemember selected from the group consisting of SEQ ID NO:2 and a proteinhaving at least 60% identity to SEQ ID NO:2.
 23. The antibody of claim22, wherein the antibody binds to SEQ ID NO:2.
 24. The antibody of claim22, wherein the antibody binds to the protein having at least 60%identity to SEQ ID NO:2.
 25. The antibody of claim 22, wherein theantibody binds to a protein selected from the group consisting of aprotein having at least 70% identity to SEQ ID NO:2, a protein having atleast 80% identity to SEQ ID NO:2, a protein having at least 90%identity to SEQ ID NO:2 and a protein having at least 95% identity toSEQ ID NO:2.
 26. The antibody of claim 22, wherein the antibody includesa monoclonal antibody.
 27. The antibody of claim 22, wherein theantibody includes a polyclonal antibody.
 28. The antibody of claim 22,wherein the antibody includes a chimeric antibody.
 29. The antibody ofclaim 22, wherein the antibody includes a human antibody.
 30. Theantibody of claim 22, wherein the antibody includes a single chainantibody.
 31. The antibody of claim 22, wherein the antibody includes anantibody fragment.
 32. The antibody of claim 28, wherein the antibodyfragment includes an Fab fragment.
 33. The antibody of claim 28, whereinthe antibody fragment includes an Fab′ fragment.
 34. The antibody ofclaim 28, wherein the antibody fragment includes an F(ab′)₂ fragment.35. The antibody of claim 22, further including a detectable label. 36.The antibody of claim 35, wherein the detectable label is selected fromthe group consisting of an enzyme, a radiolabel, a fluorescent molecule,a chemiluminescent molecule, a cofactor, an enzyme substrate, an enzymeinhibitor, and a magnetic particle.
 37. A kit comprising an isolatedantibody that binds to at least one member selected from the groupconsisting of SEQ ID NO:2 and a protein having at least 60% identity toSEQ ID NO:2.
 38. The kit of claim 37, wherein the antibody binds to SEQID NO:2.
 39. The kit of claim 37, wherein the antibody binds to theprotein having at least 60% identity to SEQ ID NO:2.
 40. The kit ofclaim 37, wherein the antibody binds to a protein selected from thegroup consisting of a protein having at least 70% identity to SEQ IDNO:2, a protein having at least 80% identity to SEQ ID NO:2, a proteinhaving at least 90% identity to SEQ ID NO:2 and a protein having atleast 95% identity to SEQ ID NO:2.
 41. The kit of claim 37, furtherincluding a labeled binding partner to the isolated antibody.
 42. Thekit of claim 37, wherein the antibody further includes a detectablelabel.